US11207375B2 - Vaccines and compositions against Streptococcus pneumoniae - Google Patents

Vaccines and compositions against Streptococcus pneumoniae Download PDF

Info

Publication number
US11207375B2
US11207375B2 US16/133,590 US201816133590A US11207375B2 US 11207375 B2 US11207375 B2 US 11207375B2 US 201816133590 A US201816133590 A US 201816133590A US 11207375 B2 US11207375 B2 US 11207375B2
Authority
US
United States
Prior art keywords
seq
polypeptide
pneumoniae
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active, expires
Application number
US16/133,590
Other versions
US20190298795A1 (en
Inventor
Todd Gierahn
Richard Malley
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Childrens Medical Center Corp
Genocea Biosciences Inc
Original Assignee
Childrens Medical Center Corp
Genocea Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Childrens Medical Center Corp, Genocea Biosciences Inc filed Critical Childrens Medical Center Corp
Priority to US16/133,590 priority Critical patent/US11207375B2/en
Assigned to CHILDREN'S MEDICAL CENTER CORPORATION reassignment CHILDREN'S MEDICAL CENTER CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MALLEY, RICHARD
Assigned to GENOCEA BIOSCIENCES, INC. reassignment GENOCEA BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GIERAHN, TODD
Publication of US20190298795A1 publication Critical patent/US20190298795A1/en
Application granted granted Critical
Publication of US11207375B2 publication Critical patent/US11207375B2/en
Active legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • S. pneumoniae is a Gram-positive encapsulated coccus that colonizes the nasopharynx in about 5-10% of healthy adults and 20-40% of healthy children. Normal colonization becomes infectious when S. pneumoniae is carried into the Eustachian tubes, nasal sinuses, lungs, bloodstream, meninges, joint spaces, bones and peritoneal cavity. S. pneumoniae has several virulence factors that enable the organism to evade the immune system.
  • Examples include a polysaccharide capsule that prevents phagocytosis by host immune cells, proteases that inhibit complement-mediated opsonization, and proteins that cause lysis of host cells.
  • the presence of complex polysaccharides forms the basis for dividing pneumococci into different serotypes. To date, 93 serotypes of S. pneumoniae have been identified.
  • a polyvalent pneumococcal vaccine PPV-23
  • the vaccine contains capsular polysaccharides (CPs) from 23 serotypes of S. pneumoniae .
  • CPs capsular polysaccharides
  • T cell independent antigens these CPs induce only short-lived antibody responses, necessitating repeated doses, which increases the risk of immunological tolerance.
  • the antibodies raised against S. pneumoniae termed anticapsular antibodies, are recognized as protective in adult and immunocompetent individuals.
  • a second S. pneumoniae vaccine includes bacterial polysaccharides from 7 S. pneumoniae strains conjugated to the diphtheria toxoid protein. This vaccine induces both B and T cell responses. However, because it only protects against 7 pneumococcal serotypes, serotype replacement can render Prevnar ineffective.
  • PPV-23 suffers from the same limitation. Serotype replacement has already been demonstrated in several clinical trials and epidemiologic studies, and raises the possibility that different formulations of the vaccines will need to be developed, presumably at even higher cost. Furthermore, both PPV-23 and Prevnar are expensive to manufacture, greatly limiting their availability in the developing world.
  • compositions need to incorporate novel or specific antigens that elicit an immune response against S. pneumoniae.
  • Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. While DNA and protein sequence information for S. pneumoniae has been known for some time, and researchers have long attempted to produce vaccines against S. pneumoniae , a major problem was how to identify protective polypeptides from among the approximately 2100 genes in the S. pneumoniae genome. The instant application presents the results of whole-genome screens designed to identify the most immunogenic proteins in the S. pneumoniae genome. Several of the hits from the screen have been shown to protect against S. pneumoniae colonization in a mouse model. Accordingly, the present disclosure provides, inter alia, certain highly effective vaccines in Streptococcus pneumoniae . The vaccines may be used therapeutically or prophylactically. The present disclosure also provides specific antigens and methods for using the antigens to elicit an immune response against S. pneumoniae.
  • the present disclosure provides, for example, a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof.
  • the present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence consisting of SEQ ID NO: 11 or an immunogenic fragment thereof.
  • the present disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence comprising SEQ ID NO: 12.
  • the instant application provides a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof.
  • This application provides, inter alia, a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein.
  • the present disclosure further provides an immunogenic composition
  • an immunogenic composition comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid sequences comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof.
  • FIG. 1 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with killed, unencapsulated whole cell S. pneumoniae , as described in Example 5.
  • the left panel shows the data in scatter format, and the right panel shows the average and standard deviation for each sample.
  • Immunization group “All 3” represents animals immunized with a combination of SP2108, SP0148, and SP1634.
  • FIG. 2 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with a combination of three proteins (SP2108, SP0148, and SP1634), as described in Example 5.
  • FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae , as described in Example 5.
  • 003 represents a control irrelevant antigen.
  • FIG. 4 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with killed, unencapsulated whole cell S. pneumoniae , as described in Example 6.
  • FIG. 5 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated by the indicated proteins and combination, as described in Example 5.
  • FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae , as described in Example 6.
  • the HSV-2 protein ICP47 with the gene name US12 (NP_044543.1, NC_001798.1; shown in the figure as 003) and ovalbumin (OVA) represent control antigens.
  • FIG. 7 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae , as described in Example 7.
  • FIG. 8 shows the number of S. pneumoniae colonies obtained from a nasal wash in BALB/c mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae , as described in Example 8.
  • S. pneumoniae vaccines that include one or more of the polypeptides or genes listed in Table 1, or variants or fragments thereof as described below.
  • the vaccine may include a polypeptide that comprises a sequence of Table 1 or a variant or immunogenic fragment thereof or a polypeptide that consists of a sequence of Table 1 or a variant or immunogenic fragment thereof.
  • SP0024 represents a hypothetical protein of 165 amino acids, containing a conserved carbonic anhydrase domain that extends from amino acid 27 to amino acid 163. Based on this consensus motif, SP0024 may be a zinc-binding protein.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0024.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 150, 125, or 100 consecutive amino acids from SP0024.
  • SP0882 is a conserved hypothetical protein of 274 amino acids. Much of the protein (amino acids 2-270) forms an esterase or lipase-like region.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0882.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids from SP0882.
  • SP0882N consists of the N-terminal 130 amino acids of SP0882, and is shown as SEQ ID NO: 3.
  • SP0882N includes a region that is particularly well conserved among different serotypes.
  • a polypeptide comprising SP0882 or SP0882N, or an immunogenic fragment of either also comprises an exogenous leader sequence.
  • the leader sequence may be, for example, the leader sequence of SP2108.
  • Two exemplary such polypeptides are SEQ ID NOS: 4 and 5.
  • the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-17, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 2-5).
  • Nucleic acid sequences encoding different variants of SP0882 are provided as SEQ ID NOS: 24-26, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) could encode these polypeptides.
  • the protein SP0148 is named “ABC transporter, substrate-binding protein”. Proteins of this class are typically extracellular proteins that interact transiently with a transmembrane protein complex. Such complexes use energy generated by ATP hydrolysis to translocate specific substrates across a cell membrane.
  • SP0148 is a 276 amino acid protein that contains a conserved PBPb (periplasmic binding protein) domain, spanning amino acids 40-246, which is typical of membrane-bound transport complexes.
  • SB0148 has a bacterial extracellular solute-binding proteins, family 3 domain which is largely co-extensive with the PBPb domain and extends from amino acid 40 to 244.
  • a vaccine or other composition comprises a truncation mutant of SP0148 comprising or lacking one or more of said domains and motifs.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0148.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids from SP0148.
  • Endogenous SP0148 comprises a putative signal sequence that may direct its secretion.
  • a variant of SP0148 that lacks the signal sequence (SEQ ID NO: 6) is used.
  • the polypeptide of SEQ ID NO: 6 is encoded by the nucleic acid of SEQ ID NO: 27, although other nucleic acid sequences (including codon-optimized sequences) may be used.
  • SEQ ID NO: 28 encodes the full length sequence of SP0148 used in the screens herein.
  • the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 18-19, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 6 or 7).
  • SP1072 also known as dnaG, is a DNA primase enzyme that catalyzes formation of an RNA primer which allows DNA polymerase to initiate DNA replication.
  • a protein of 586 amino acids, SP1072 contains several conserved motifs. Beginning at the N-terminus, amino acids 2-96 form a zinc finger domain, the DNA primase catalytic core spans amino acids 122-250, and a highly conserved topoisomerase-primase (TORPIM) nucleotidyl transferase/hydrolase domain region extends from amino acid 258 to 330.
  • a vaccine or other composition comprises a truncation mutant of SP1072 comprising or lacking one or more of said domains and motifs.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected form SP1072.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP1072.
  • the polypeptide SP2108 is 423 amino acids in length and is alternatively known as MalX, maltose/maltodextrin ABC transporter, or maltose/maltodextrin-binding protein. Much of the protein (amino acids 3-423) is classified as a MalE (Maltose-binding periplasmic) domain.
  • SP2108 contains a signal sequence that directs its secretion.
  • a vaccine or other composition comprises a truncation mutant of SP2108 comprising one or more of said domains and motifs.
  • compositions and methods herein call for the use of an SP2108 variant that lacks the signal sequence.
  • This variant is represented by polypeptide sequence SEQ ID NO: 10 and may be encoded by, for example, a nucleic acid according to SEQ ID NO: 29.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP2108.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP2108.
  • the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 20-21, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 9 or 10).
  • SP0641 is also known as PrtA, a cell wall-associated serine protease.
  • Full-length SP0641 contains a number of conserved motifs: the PA_2 motif, extending between amino acids 485 and 597, which may form a protein binding surface; the Fn3-like domain (amino acids 800-939); and two predicted catalytic domains of the SS8 C5a type located at amino acids 226-449 and 639-777.
  • a vaccine or other composition comprises a truncation mutant of SP0641 comprising or lacking one or more of said domains and motifs.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0641.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 consecutive amino acids from SP0641.
  • SP0641N SEQ ID NO: 13
  • SP0641N Roughly adjacent to SP0641N (and corresponding to amino acids 686-1333 of SP0641) lies the 648 residue region captured by the truncation variant SP0641M (SEQ ID NO: 11).
  • SEQ ID NOS: 30 and 31 display the DNA sequences of SP0641M and SP0641N, respectively, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) could encode SP0641.
  • Polypeptides homologous to the polypeptides of Tables 1 and 2 may also be used in the compositions and methods disclosed herein.
  • Individual strains of S. pneumoniae contain numerous mutations relative to each other, and some of these result different protein sequences between the different strains.
  • One of skill in the art may readily substitute an amino acid sequence, or a portion thereof, with the homologous amino acid sequence from a different S. pneumoniae strain.
  • this application provides immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the polypeptides of Tables 1 and 2 or an immunogenic fragment thereof. Serotypic variation may be used to design such variants of the polypeptides of Tables 1 and 2.
  • the vaccine compositions herein comprise a fragment of a protein of Table 1 or 2 (for example, fragments of SP0024, SP0882, SP0882N, 0SP148 with or without a signal sequence, SP1072, SP1028 with or without a signal sequence, SP0641, SP0641M, or SP0641N).
  • this application provides truncation mutants that are close in size to the polypeptide of Table 1 or 2 (for example, one of SEQ ID NOS: 1-13). For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from one or both termini. Internal deletions, e.g., of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.
  • the vaccine formulation comprises one or more polypeptides having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-21.
  • the fragment is a truncated fragment of any of SEQ ID NOS: 14-21 wherein from 1-5, 1-10, or 1-20 amino acid residues are removed from the N-terminus, C-terminus, or both.
  • the fragment is a truncated fragment of any of SEQ ID NOS: 14-21 wherein from 1-10 amino acid residues are removed from the N-terminus, C-terminus, or both. For instance, 10 amino acid residues may be removed from each of the N-terminus and C-terminus resulting in a protein with 20 amino acid residues removed.
  • this application also provides immunogenic compositions that include one or more of the polypeptides or genes listed in Table 1 and/or Table 2, or variants or fragments thereof as described herein.
  • the DNA and protein sequence of each gene and protein may be found by searching for the Locus Tag in the publicly available database, Entrez Gene, as described above.
  • polypeptides present in compounds of the invention are immunogenic, either alone or as a variant, which includes polypeptides fused to another polypeptide or mixed with or complexed to an adjuvant.
  • variants also include sequences with less than 100% sequence identity, as described herein.
  • an antigen of Table 1 or 2 is provided as a full length polypeptide.
  • fragments, precursors and analogs that have an appropriate immunogenicity.
  • polypeptides may be immunogenic in mammals, for example mice, guinea pigs, or humans.
  • An immunogenic polypeptide is typically one capable of raising a significant immune response in an assay or in a subject.
  • an immunogenic polypeptide may increase the amount of IL-17 produced by T cells.
  • the IL-17 assay described in Examples 1-4 is an example of an assay that may be used to identify an immunogenic polypeptide.
  • an immunogenic polypeptide may (i) induce production of antibodies, e.g., neutralizing antibodies, that bind to the polypeptide (ii) induce T H 1 immunity.
  • an immunogenic polypeptide causes the production of a detectable amount of antibody specific to that antigen.
  • polypeptides have less than 20%, 30%, 40%, 50%, 60% or 70% identity to human autoantigens and/or gut commensal bacteria (e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia and Lactobacillus species).
  • human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.
  • the present disclosure provides, for example, a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof.
  • the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof.
  • the term “different” signifies that each of said two peptides originates from a different sequence selected from SEQ ID NOS: 1-13.
  • the vaccine formulation may also comprise one or more polypeptides having an amino acid sequence consisting of any of SEQ ID NOS: 1-11.
  • the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragment thereof. (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or an immunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 or an immunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13 or an immunogenic fragment thereof. Examples of such combinations are listed below:
  • the vaccine formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10.
  • the vaccine formulation comprises at least three polypeptides, each polypeptide belonging to a different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragment thereof.
  • SEQ ID NO: 1 SEQ ID NO: 2, and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 3, and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 4; and SEQ ID NO: 6
  • SEQ ID NO: 1 SEQ ID NO: 4; and SEQ ID NO: 7
  • SEQ ID NO: 1 SEQ ID NO: 4; and SEQ ID NO: 8
  • SEQ ID NO: 1 SEQ ID NO: 4; and SEQ ID NO: 10
  • SEQ ID NO: 1 SEQ ID NO: 4; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 6
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 7
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 8
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 10
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 5; and SEQ ID NO: 13
  • SEQ ID NO: 1 SEQ ID NO: 6; and SEQ ID NO: 8
  • SEQ ID NO: 1 SEQ ID NO: 6; and SEQ ID NO: 10
  • SEQ ID NO: 1 SEQ ID NO: 6; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 6; and SEQ ID NO: 13
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 8
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 9
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 10
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 11
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 7; and SEQ ID NO: 13
  • SEQ ID NO: 1 SEQ ID NO: 8; and SEQ ID NO: 9
  • SEQ ID NO: 1 SEQ ID NO: 8; and SEQ ID NO: 10
  • SEQ ID NO: 1 SEQ ID NO: 8; and SEQ ID NO: 11
  • SEQ ID NO: 1 SEQ ID NO: 8; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 8; and SEQ ID NO: 13
  • SEQ ID NO: 1 SEQ ID NO: 9; and SEQ ID NO: 11
  • SEQ ID NO: 1 SEQ ID NO: 9; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 9; and SEQ ID NO: 13
  • SEQ ID NO: 1 SEQ ID NO: 10; and SEQ ID NO: 11
  • SEQ ID NO: 1 SEQ ID NO: 10; and SEQ ID NO: 12
  • SEQ ID NO: 1 SEQ ID NO: 10; and SEQ ID NO: 13
  • SEQ ID NO: 2 SEQ ID NO: 6; and SEQ ID NO: 8
  • SEQ ID NO: 2 SEQ ID NO: 6; and SEQ ID NO: 10
  • SEQ ID NO: 2 SEQ ID NO: 6; and SEQ ID NO: 12
  • SEQ ID NO: 2 SEQ ID NO: 7; and SEQ ID NO: 8
  • SEQ ID NO: 2 SEQ ID NO: 7; and SEQ ID NO: 10
  • SEQ ID NO: 2 SEQ ID NO: 7; and SEQ ID NO: 12
  • SEQ ID NO: 2 SEQ ID NO: 8; and SEQ ID NO: 10
  • SEQ ID NO: 2 SEQ ID NO: 8; and SEQ ID NO: 12
  • SEQ ID NO: 2 SEQ ID NO: 9; and SEQ ID NO: 12
  • SEQ ID NO: 2 SEQ ID NO: 10; and SEQ ID NO: 11
  • SEQ ID NO: 2 SEQ ID NO: 10; and SEQ ID NO: 12
  • SEQ ID NO: 3 SEQ ID NO: 6; and SEQ ID NO: 8
  • SEQ ID NO: 3 SEQ ID NO: 6; and SEQ ID NO: 10
  • SEQ ID NO: 3 SEQ ID NO: 6; and SEQ ID NO: 12
  • SEQ ID NO: 3 SEQ ID NO: 7; and SEQ ID NO: 8
  • SEQ ID NO: 3 SEQ ID NO: 7; and SEQ ID NO: 10
  • SEQ ID NO: 3 SEQ ID NO: 7; and SEQ ID NO: 12
  • SEQ ID NO: 3 SEQ ID NO: 8; and SEQ ID NO: 10
  • SEQ ID NO: 3 SEQ ID NO: 8; and SEQ ID NO: 12
  • SEQ ID NO: 3 SEQ ID NO: 9; and SEQ ID NO: 12
  • SEQ ID NO: 3 SEQ ID NO: 10; and SEQ ID NO: 11
  • SEQ ID NO: 3 SEQ ID NO: 10; and SEQ ID NO: 12
  • SEQ ID NO: 4 SEQ ID NO: 6; and SEQ ID NO: 12
  • SEQ ID NO: 4 SEQ ID NO: 7; and SEQ ID NO: 10
  • SEQ ID NO: 4 SEQ ID NO: 7; and SEQ ID NO: 12
  • SEQ ID NO: 4 SEQ ID NO: 8; and SEQ ID NO: 10
  • SEQ ID NO: 4 SEQ ID NO: 8; and SEQ ID NO: 12
  • SEQ ID NO: 4 SEQ ID NO: 9; and SEQ ID NO: 12
  • SEQ ID NO: 4 SEQ ID NO: 10; and SEQ ID NO: 12
  • SEQ ID NO: 6 SEQ ID NO: 10; and SEQ ID NO: 12
  • the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof.
  • the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i)-(iii): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragment thereof. Examples of such combinations are listed below:
  • a vaccine formulation comprising one or more of SEQ ID NOS: 14-21 further comprises a polypeptide having an amino acid sequence comprising any of SEQ ID NOS: 1-13.
  • the vaccine formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof.
  • the vaccine formulation comprises three of (i)-(iii): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragment thereof. Examples of such combinations are listed below:
  • a polypeptide may comprise one or more immunogenic portions and one or more non-immunogenic portions.
  • the immunogenic portions may be identified by various methods, including protein microarrays, ELISPOT/ELISA techniques, and/or specific assays on different deletion mutants (e.g., fragments) of the polypeptide in question.
  • Immunogenic portions may also be identified by computer algorithms. Some such algorithms, like EpiMatrix (produced by EpiVax), use a computational matrix approach.
  • An immunogenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by algorithm.
  • Peptides identified by the tools described above include the following:
  • this application provides an immunogenic fragment of an antigen described herein.
  • the fragments in some instances, are close in size to the full-length polypeptide or the polypeptide of Table 1 or 2. For example, they may lack at most one, two, three, four, five, ten, or twenty amino acids from one or both termini.
  • the polypeptide is 100-500 amino acids in length, or 150-450, or 200-400, or 250-250 amino acids in length. In some embodiments, the polypeptide is 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acids in length.
  • fragments described above or sub-fragments thereof preferably have one of the biological activities described below, such as increasing the amount of IL-17 released by at least 1.5 fold or 2 fold or more (e.g., either as an absolute measure or relative to an immunologically inactive protein such as ovalbumin).
  • a fragment may be used as the polypeptide in the vaccines described herein or may be fused to another protein, protein fragment or a polypeptide.
  • the fragment is a truncated fragment of any of SEQ ID NOS: 1-13 having from 1-5, 1-10, or 1-20 amino acid residues removed from the N-terminus, C-terminus, or both.
  • the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus.
  • this application provides immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a polypeptide of Table 1 or 2.
  • the present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof.
  • the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof.
  • one or more, e.g. two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof are provided in a mixture.
  • two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof are covalently bound to each other, e.g. as a fusion protein.
  • the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Table 1. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Tables 1 or 2.
  • vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NO: 1-13. In certain such embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence consisting of any of SEQ ID NO: 1-13. In some embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising (or consisting of) any of the amino acid sequences of the polypeptides of Tables 1 and 2.
  • Substantially in this context, refers to less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2, or even less than 1% of the other S. pneumoniae polypeptides.
  • the vaccine composition induces a T H 17 cell response at least 1.5-fold than that induced by a control irrelevant antigen (such as the HSV-2 protein ICP47 with the gene name US12) after contacting T H 17 cells.
  • a control irrelevant antigen such as the HSV-2 protein ICP47 with the gene name US12
  • the vaccine formulation inhibits infection by S. pneumoniae in an uninfected subject.
  • the vaccine formulation inhibits S. pneumoniae colonization in an individual.
  • the vaccine formulation inhibits S. pneumoniae symptoms.
  • this application provides nucleic acids encoding one or more of the polypeptides described above, such as DNA, RNA, or an analog thereof.
  • the underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention.
  • the DNA sequence may be codon-optimized to improve expression in a host such as E. coli , an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
  • this application provides nucleic acids (such as DNA. RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a gene in Table 1 or 2, or a variant or portion of said gene.
  • the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000 nucleotides in length.
  • the nucleic acids may be used, for example, for recombinant production of the polypeptides of Tables 1 and 2, or immunogenic fragments thereof.
  • the vaccine or immunogenic composition may comprise fusion proteins and/or fusion DNA constructs.
  • the polypeptides described herein may be used without modification.
  • the smaller polypeptides can be conjugated to an appropriate immunogenic carrier such as tetanus toxoid, pneumolysin keyhole limpet hemocyanin or the like.
  • the vaccine formulation comprises at least one lipidated polypeptide.
  • Conjugation may be direct or indirect (e.g., via a linker).
  • a construct may comprise a gene or protein from Table 1 or 2 or an immunogenic fragment or variant thereof and a tag.
  • a tag may be N-terminal or C-terminal.
  • tags may be added to the nucleic acid or polypeptide to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein or nucleic acid.
  • a purification tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification.
  • the polypeptide is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag.
  • the fused portion is short. This, in some instances, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of the polypeptide of Table 1 or 2.
  • the present disclosure also provides pharmaceutical compositions containing immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides together with a pharmaceutical carrier.
  • Antigens from S. pneumoniae were identified by screening immune cells from mice infected with S. pneumonia , or from healthy human donors. The human donors had presumably been exposed to S. pneumoniae at some point during their lifetimes, because S. pneumoniae is a very common disease and colonizing pathogen. Briefly, a library of S. pneumoniae antigens was expressed in bacteria and mixed with antigen presenting cells (APCs). The APCs, in turn, presented S. pneumoniae -derived polypeptides to lymphocytes that had been isolated from mice or from human donors. Lymphocyte responses were assayed for reactivity to S.
  • APCs antigen presenting cells
  • compositions of the S. pneumoniae antigens that elicit a strong immune response in immunized or infected mice or humans for counteracting infection by S. pneumoniae.
  • Tables 1 and 2 list the protein sequence and corresponding nucleotide sequence for S. pneumoniae antigens identified according to the screening methods described herein.
  • the antigens were identified in screens of mouse and human T cells. In the screens of mouse T cells, the identified antigens were subjected to at least two rounds of screening: a genome-wide round to identify pools of 4 antigens that elicited an immune response, followed by a deconvolution round to individually test and identify single antigens that elicited an immune response from a pool identified in the genome-wide round. In contrast, in the screens of human T cells, two different sets of antigen pools were created, such that a polypeptide was combined with different polypeptides between the first and second pools.
  • compositions according to this disclosure may include one or two or more of the genes listed in Table 1 or 2, or the corresponding gene products.
  • An immunogenic composition may also comprise portions of said Streptococcus polypeptides, for example deletion mutants, truncation mutants, oligonucleotides, and peptide fragments.
  • the portions of said polypeptides are immunogenic.
  • the immunogenicity of a portion of a protein is readily determined using the same assays that are used to determine the immunogenicity of the full-length protein.
  • the portion of the polypeptide has substantially the same immunogenicity as the full-length proteins.
  • the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less than that of the full-length protein (e.g., polypeptides of Tables 1 and 2).
  • the peptide fragments may be, for example, linear, circular, or branched.
  • Some embodiments of the vaccine formulations and immunogenic compositions described herein include an immunogenic polypeptide (e.g., a polypeptide of Table 1 or 2) that contains a membrane translocating sequence (MTS), to facilitate introduction of the polypeptide into the mammalian cell and subsequent stimulation of the cell-mediated immune response.
  • an immunogenic polypeptide e.g., a polypeptide of Table 1 or 2
  • MTS membrane translocating sequence
  • Exemplary membrane translocating sequences include hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of ⁇ -synuclein, ⁇ -synuclein, or ⁇ -synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin ⁇ 3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680 and 6,248,558.
  • an antigen e.g., a polypeptide of Table 1 or 2
  • another molecule is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioabailability, or immunogenicity of the antigen.
  • Molecules that may be covalently bound to the antigen include a carbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA.
  • PEG poly(ethylene glycol)
  • PEG polysialic acid
  • N-propionylated polysialic acid nucleic acids
  • polysaccharides and PLGA.
  • PEG poly(ethylene glycol)
  • PEG chains can be linear, branched, or with comb or star geometries.
  • the naturally produced form of a protein is covalently bound to a moeity that stimulates the immune system.
  • a moeity is a lipid moeity.
  • lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR2, and activate the innate immune system.
  • TLR Toll-like receptor
  • an antibody preparation generated against an antigenic composition e.g., one of the proteins listed in Table 1 or 2 or an immunogenic fragment thereof.
  • an antigenic composition e.g., one of the proteins listed in Table 1 or 2 or an immunogenic fragment thereof.
  • this disclosure provides combinations of two, three, four, or five antibodies each recognizing a different protein of Table 1 or 2. Any of a variety of antibodies are included.
  • Such antibodies include, e.g., polyclonal, monoclonal, recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof, etc.
  • the antibodies can be of any isotype, e.g., IgG, various IgG isotypes such as IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, etc.; and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like.
  • Fab molecules are expressed and assembled in a genetically transformed host like E. coli .
  • a lambda vector system is available thus to express a population of Fab's with a potential diversity equal to or exceeding that of subject generating the predecessor antibody. See Huse et al. (1989), Science 246, 1275-81.
  • the vaccine or immunogenic composition comprises an antigen and one or more of the following: an adjuvant, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and an antibody specific to said antigen.
  • the vaccine formulations and immunogenic compositions described herein may include an adjuvant.
  • Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et al., Curr. HIV Res. 1:309-20, 2003).
  • Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), which may be, for example, particles and/or matrices, and liposomes.
  • ISCOMs immune-stimulating complexes
  • immunostimulatory adjuvants are sometimes derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
  • PAMP pathogen associated molecular patterns
  • LPS lipopolysaccharides
  • MPL monophosphoryl lipid
  • CpG-containing DNA which activate cells of the innate immune system.
  • adjuvants may be classified as organic and inorganic.
  • Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines.
  • Organic adjuvants comprise organic molecules including macromolecules.
  • An example of an organic adjuvant is cholera toxin.
  • Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the activation of T H 1 cells or T H 2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
  • the adjuvant induces the activation of T H 17 cells. It may promote the T H 17 cells to secrete IL-17.
  • an adjuvant that induces the activation of T H 17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL-17 levels secreted by two populations of cells: (1) cells treated with the adjuvant and a polypeptide known to induce T H 17 activation, and (2) cells treated with the adjuvant and an irrelevant (control) polypeptide.
  • An adjuvant that induces the activation of T H 17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2).
  • IL-17 may be measured, for example, by ELISA or Western blot.
  • Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli , or ETEC) activate a T 11 17 response.
  • the adjuvant is a toxin. Cholera toxin was successfully used in the mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1 (see Examples 5-8).
  • One form of labile toxin is produced by Intercell.
  • Mutant derivates of labile toxin that are active as adjuvants but significantly less toxic can be used as well.
  • Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity.
  • Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al., “Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants” PNAS Vol. 92, pp.
  • LTK63 Bacilliams et al., “Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease” The Journal of Immunology, 2004, 173: 7435-7443), LT-G192 (Douce et al. “Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants” Infect Immun.
  • LTR72 Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.” J Exp Med. 1998 Apr. 6:187(7):1123-32).
  • the adjuvant comprises a VLP (virus-like particle).
  • VLP virus-like particle
  • Alphavirus replicons induces the activation of T H 17 cells using alphavirus and is produced by Alphavax.
  • alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Pat. Nos. 5,643,576 and 6,783,939.
  • a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.
  • TLRs toll-like receptors
  • LPS lipopolysaccharide
  • TLR4 agonists synthetic TLR agonists
  • TLR4 agonists synthetic TLR agonists
  • E6020 synthetic phospholipid dimer
  • TLR agonists include TLR4 agonists
  • TLR4 agonists have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids. Inc., and Sigma Aldrich.
  • Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA).
  • TDM Trehalose Dicoynomycolate
  • DDA dioctadecyldimethylammonium bromide
  • R848 resiquimod
  • the adjuvant is or comprises a saponin.
  • the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree.
  • a saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity.
  • Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.
  • ISCOMs immunostimulating complexes
  • ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline).
  • an ISCOM is assembled together with a polypeptide or nucleic acid of interest.
  • different saponin fractions may be used in different ratios.
  • the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions).
  • Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A:30-5 C, such as 70 A:30 C to 75 A:5 C, 75 A:5 C to 80 A:20 C, 80 A:20 C to 85 A:15 C, 85 A:15 C to 90 A:10 C, 90 A:10 C to 95 A:5 C, or 95 A:5 C to 99 A:1 C.
  • combinations of adjuvants are used.
  • Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.
  • Adjuvants may be covalently bound to antigens.
  • the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al., Cancer Res 2005; 65(11), pp 4947-4954).
  • a polypeptide described herein is presented in the context of the trivalent S.
  • pneumoniae Pneumococcal surface adhesin A pneumolysin derivative carrying three amino acid substitutions (W433F, D385N, and C428G) which render the molecule nontoxic but do not interfere with TLR4-mediated inflammatory properties-cell wall polysaccharide (PsaA:PdT-CPs) conjugate system described in Lu et al. (“Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide.” Infect Immun. 2009 May; 77(5):2076-83).
  • the conjugate system is “a fusion protein of PsaA with the pneumolysin nontoxic derivative PdT and then coupled CPs to the fusion protein”.
  • one or more polypeptides described herein is used in place of PsaA in the trivalent conjugate.
  • the trivalent conjugate system typically includes alum and is usually administered parenterally.
  • Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.
  • the same adjuvant or mixture of adjuvants is present in each dose of a vaccine.
  • an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (i.e., booster shots).
  • a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses.
  • the adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
  • a vaccine formulation or immunogenic composition may include one or more additional components.
  • the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
  • stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or mono
  • the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid.
  • the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.
  • PBS phosphate buffered saline
  • the vaccine formulation or immunogenic composition includes one or more surfactants such as polysorbate 80 (TWEEN® 80), Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-(1,1,3,3 Tetramethylbutyl)phenyl-polyethylene glycol (TRITON® X-100); Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN® 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL®).
  • a surfactant can be ionic or nonionic.
  • the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
  • a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
  • the vaccine formulation or immunogenic composition is a controlled release formulation.
  • the vaccine comprises one of the nucleic acids disclosed herein or a nucleic acid corresponding to one of the polypeptides described herein.
  • a nucleic acid vaccine When a nucleic acid vaccine is administered to a patient, the corresponding gene product (such as a desired antigen) is produced in the patient's body.
  • nucleic acid vaccine vectors that include optimized recombinant polynucleotides can be delivered to a mammal (including humans) to induce a therapeutic or prophylactic immune response.
  • the nucleic acid may be, for example, DNA, RNA, or a synthetic nucleic acid.
  • the nucleic acid may be single stranded or double stranded.
  • Nucleic acid vaccine vectors e.g., adenoviruses, liposomes, papillomaviruses, retroviruses, etc.
  • the nucleic acid vaccines can be formulated as pharmaceutical compositions for administration in any suitable manner, including parenteral administration.
  • the physician evaluates vector toxicities, progression of the disease, and the production of anti-vector antibodies, if any.
  • the dose equivalent of a naked nucleic acid from a vector is from about 1 ⁇ g to 1 mg for a typical 70 kilogram patient, and doses of vectors used to deliver the nucleic acid are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • Administration can be accomplished via single or divided doses.
  • the toxicity and therapeutic efficacy of the nucleic acid vaccine vectors can be determined using standard pharmaceutical procedures in cell cultures or experimental animals.
  • a nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, or a combination thereof.
  • the vaccine comprises one or more cloning or expression vectors; for instance, the vaccine may comprise a plurality of expression vectors each capable of autonomous expression of a nucleotide coding region in a mammalian cell to produce at least one immunogenic polypeptide.
  • An expression vector often includes a eukaryotic promoter sequence, such as the nucleotide sequence of a strong eukaryotic promoter, operably linked to one or more coding regions.
  • the compositions and methods herein may involve the use of any particular eukaryotic promoter, and a wide variety are known; such as a CMV or RSV promoter.
  • the promoter can be heterologous with respect to the host cell.
  • the promoter used may be a constitutive promoter.
  • a vector useful in the present compositions and methods can be circular or linear, single-stranded or double stranded and can be a plasmid, cosmid, or episome.
  • each nucleotide coding region is on a separate vector; however, it is to be understood that one or more coding regions can be present on a single vector, and these coding regions can be under the control of a single or multiple promoters.
  • nucleic acid vaccines Numerous plasmids may be used for the production of nucleic acid vaccines. Suitable embodiments of the nucleic acid vaccine employ constructs using the plasmids VR1012 (Vical Inc., San Diego Calif.), pCMVI.UBF3/2 (S. Johnston. University of Texas) or pcDNA3.1 (InVitrogen Corporation, Carlsbad, Calif.) as the vector.
  • the vector construct can contain immunostimulatory sequences (ISS), such as unmethylated dCpG motifs, that stimulate the animal's immune system.
  • the nucleic acid vaccine can also encode a fusion product containing the immunogenic polypeptide.
  • Plasmid DNA can also be delivered using attenuated bacteria as delivery system, a method that is suitable for DNA vaccines that are administered orally. Bacteria are transformed with an independently replicating plasmid, which becomes released into the host cell cytoplasm following the death of the attenuated bacterium in the host cell.
  • nucleic acid is a vector, such as a plasmid, that is capable of autologous expression of the nucleotide sequence encoding the immunogenic polypeptide.
  • the S. pneumoniae vaccines described herein may be used for prophylactic and/or therapeutic treatment of S. pneumoniae . Accordingly, this application provides a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein. In some aspects, the method inhibits S. pneumoniae colonization in an individual. In some aspects, the method inhibits S. pneumoniae symptoms.
  • the subject receiving the vaccination may be a male or a female, and may be a child or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal.
  • the vaccine is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae , for example by protecting against colonization, the first and necessary step in disease.
  • the method inhibits infection by S. pneumoniae in a noncolonized or uninfected subject.
  • the method may reduce the duration of colonization in an individual that is already colonized.
  • the vaccine compositions of the invention confer protective immunity, allowing a vaccinated individual to exhibit delayed onset of symptoms or reduced severity of symptoms, as the result of his or her exposure to the vaccine.
  • the reduction in severity of symptoms is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%.
  • vaccinated individuals may display no symptoms upon contact with S. pneumoniae , do not become colonized by S. pneumoniae , or both.
  • Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts.
  • IgA secretory IgA
  • the sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body.
  • Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum.
  • Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies.
  • cellular immunity may be mediated by T H 1 or T H 17 cells.
  • a vaccine formulation as described herein e.g., a composition comprising one or more polypeptides from Table 1 or 2, or nucleic acids encoding the polypeptides, or antibodies reactive with the polypeptides
  • a vaccine formulation as described herein is administered to patients that are immunocompromised.
  • An immunocompromising condition arising from a medical treatment is likely to expose the individual in question to a higher risk of infection with S. pneumoniae . It is possible to treat an infection prophylactically in an individual having the immunocompromised condition before or during treatments known to compromise immune function.
  • an antigenic composition e.g., two or more antigens from Table 1 or 2, or nucleic acids encoding the antigens
  • an antigenic composition e.g., two or more antigens from Table 1 or 2, or nucleic acids encoding the antigens
  • antibodies reactive to two or more antigens from Table 1 or 2 before or during a treatment known to compromise immune function
  • the individual contract an S. pneumoniae infection e.g., following a treatment leading to an immunocompromised condition it is also possible to treat the infection by administering to the individual an antigen composition.
  • the following groups are at increased risk of pneumococcal disease or its complications, and therefore it is advantageous for subjects falling into one or more of these groups to receive a vaccine formulation described herein: children, especially those from 1 month to 5 years old or 2 months to 2 years old; children who are at least 2 years of age with asplenia, splenic dysfunction or sickle-cell disease; children who are at least 2 years of age with nephrotic syndrome, chronic cerebrospinal fluid leak, HIV infection or other conditions associated with immunosuppression.
  • At least one dose of the pneumococcal antigen composition is given to adults in the following groups at increased risk of pneumococcal disease or its complications: all persons 65 years of age; adults with asplenia, splenic dysfunction or sickle-cell disease; adults with the following conditions: chronic cardiorespiratory disease, cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDS and other conditions associated with immunosuppression (Hodgkin's disease, lymphoma, multiple myeloma, immunosuppression for organ transplantation), individuals with cochlear implants; individuals with long-term health problems such as heart disease and lung disease, as well as individuals who are taking any drug or treatment that lowers the body's resistance to infection, such as long-term steroids, certain cancer drugs, radiation therapy; Alaskan natives and certain Native American populations.
  • immunosuppression Hodgkin's disease, lymphoma, multiple myeloma
  • the vaccine may be administered to a patient suffering from S. pneumoniae infection, in an amount sufficient to treat the patient. Treating the patient, in this case, refers to reducing symptoms, bacterial load, or both of S. pneumoniae in an infected individual. In some embodiments, treating the patient refers to reducing the duration of symptoms or reducing the intensity of symptoms. In some embodiments, the vaccine reduces transmissibility of S. pneumoniae from the vaccinated patient. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%.
  • the vaccine is administered to an individual post-infection.
  • the vaccine may be administered shortly after infection, e.g. before symptoms manifest, or may be administered during or after manifestation of symptoms.
  • a therapeutic S. pneumoniae vaccine can reduce the intensity and/or duration symptoms of the various indications of S. pneumoniae infection.
  • a S. pneumoniae infection can take many forms. In some cases, an infected patient develops pneumonia, acute sinusitis, otitis media (ear infection), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.
  • the efficacy of vaccination with the vaccines disclosed herein may be determined in a number of ways, in addition to the clinical outcomes described above.
  • IL-17 levels particularly IL-17A
  • the IL-17 levels may be compared to IL-17 levels in the same subject before vaccination.
  • Increased IL-17 (e.g., IL-17A) levels such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine.
  • IL-17 e.g., IL-17A
  • neutrophils in the presence of T cells or antibodies from the patient for pneumococcal killing.
  • Increased pneumococcal killing such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine.
  • T H 17 cell activation such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the vaccine.
  • levels of an antibody specific to the vaccine such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased vaccine efficacy.
  • two or more of these assays are used. For example, one may measure IL-17 levels and the levels of vaccine-specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of pneumococcal infection in vaccinated individuals compared to unvaccinated individuals.
  • Vaccine efficacy may also be assayed in various model systems such as the mouse model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering the test vaccine to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of S. pneumoniae . In some cases, the challenge dose is sufficient to cause S. pneumoniae colonization (especially nasal colonization) in an unvaccinated animal, and in some cases the challenge dose is sufficient to cause a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals. Examples 5 and 6 show the efficacy of polypeptides of Table 1 in inhibiting S. pneumoniae nasal colonization in the mouse model.
  • compositions of the present disclosure are designed to elicit an immune response against S. pneumoniae .
  • Compositions described herein e.g., ones comprising one or more polypeptides of Table 1 or 2, or nucleic acids encoding the polypeptides
  • the composition stimulates a T H 1-biased CD4+ T cell response, a T H 17-biased CD4+ T cell response, or a CD8+ T cell response; in the case of a single component composition, the composition may stimulate an antibody response, a T H 1-biased CD4+ T cell response, T H 17-biased CD4+ T cell response, and/or a CD8+ T cell response.
  • the composition (e.g., one comprising one or more polypeptides of Table 1 or 2, or nucleic acids encoding the polypeptides, or antibodies reactive with the peptides) includes a cytokine or nucleotide coding region encoding a cytokine such as IL-17, to provide additional stimulation to the immune system of the mammal.
  • the composition comprises a cytokine such as IL-17.
  • a T H 17 cell response is beneficial in mounting an immune response to the compositions disclosed herein, e.g., ones comprising one or more polypeptides of Table 1 or 2.
  • an active T H 17 response is beneficial in clearing a pneumococcal infection.
  • mice lacking the IL-17A receptor show decreased whole cell vaccine-based protection from a pneumococcal challenge (Lu et al., “Interleukin-17A mediates acquired immunity to pneumococcal colonization.” PLoS Pathog. 2008 Sep. 19; 4(9)).
  • the same authors showed that the response level of IL-17A was increased in mice treated with a whole-cell vaccine.
  • a method of increasing IL-17 production by administering the compositions described herein (e.g., ones comprising one or more polypeptides of Table 1 or 2) to a subject.
  • this application provides a method of activating T H 7 cells by administering said compositions to a subject.
  • increased IL-17A levels result in increased pneumococcal killing by neutrophils or neutrophil-like cells, for instance by inducing recruitment and activation of neutrophils of neutrophil-like cells.
  • this pneumococcal killing is independent of antibodies and complement.
  • specific antibody production and complement activation may be useful additional mechanisms that contribute to clearing of a pneumococcal infection.
  • Immunogenic compositions containing immunogenic polypeptides or polynucleotides encoding immunogenic polypeptides together with a pharmaceutical carrier are also provided.
  • the immunogenic composition comprises one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, such as one or more nucleic acids selected from SEQ ID Nos. 24-31.
  • these nucleic acids are expressed in the immunized individual, producing the encoded S. pneumoniae antigens, and the S. pneumoniae antigens so produced can produce an immunostimulatory effect in the immunized individual.
  • Such a nucleic acid-containing immunostimulatory composition may comprise, for example, an origin of replication, and a promoter that drives expression of one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13.
  • Such a composition may also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, and a polyadenylation/transcriptional termination sequence.
  • the nucleic acid is DNA.
  • Detection kits can be simple enough to be set up in any local hospital laboratory, and the antibodies and antigen-binding portions thereof can readily be made available to all hospitals treating patients with or at risk for S. pneumoniae infection.
  • patient refers to an individual (such as a human) that either has an S. pneumoniae infection or has the potential to contract an S. pneumoniae infection.
  • a patient may be an individual (such as a human) that has an S. pneumoniae infection, has the potential to contract an S. pneumoniae infection, who has recovered from S. pneumoniae infection, and/or an individual whose infection status is unknown.
  • the instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more S. pneumoniae -specific antibodies or antigen-binding portions thereof under conditions that allow for binding of the antibody or antigen-binding portion to an epitope of S. pneumoniae ; where binding indicates the presence of S. pneumoniae in the sample.
  • the binding to the biological sample is compared to binding of the same antibody to a negative control tissue, wherein if the biological sample shows the presence of S. pneumoniae as compared to the negative control tissue, the patient is identified as likely having a S. pneumoniae infection.
  • binding of one antibody indicates the presence of S. pneumoniae ; in other cases, the binding of two or more antibodies indicates the presence of S. pneumoniae .
  • the aforementioned test may be appropriately adjusted to detect other bacterial infections, for instance by using an antibody immunoreactive a homolog (from another bacterial species) of one of the proteins described in Table 1.
  • the antibodies raised against a S. pneumoniae protein in Table 1 or 2 will also bind the homolog in another Streptococcus species, especially if the homologs have a high percentage sequence identity.
  • an antigen of Table 1 or 2 may be used to detect anti- S. pneumoniae antibodies in an individual.
  • the instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more S. pneumoniae -specific antigens selected from Table 1 or 2 or portions thereof under conditions that allow for binding of the antigen (or portion thereof) to any host antibodies present in the sample; where binding indicates the presence of anti- S. pneumoniae antibodies in the sample.
  • the binding to the biological sample is compared to binding of the same antigen to a negative control tissue, wherein if the biological sample shows the presence of anti- S.
  • detecting one antibody indicates a current or past infection with S. pneumoniae ; in other cases, detecting two or more antibodies indicates a current or past infection with S. pneumoniae .
  • the aforementioned test may be appropriately adjusted to detect other bacterial infections, for instance by using a homolog (from another bacterial species (e.g., a Streptococcal species) of the proteins described in Table 1.
  • the immune cell response of a mammalian cell may be quantified ex vivo.
  • a method for such quantification comprises administering the compositions herein disclosed to a mammalian T cell ex vivo, and quantifying the change in cytokine production of the mammalian T cell in response to the composition.
  • the cytokine may be, for example, IL-17.
  • the binding of an S. pneumoniae antibody to an antigen may be measured using any appropriate method. Such methods include ELISA (enzyme-linked immunosorbent assay), Western blotting, competition assay, and spot-blot.
  • the detection step may be, for instance, chemiluminescent, fluorescent, or colorimetric.
  • One suitable method for measuring antibody-protein binding is the Luminex xMAP system, where peptides are bound to a dye-containing microsphere. Certain systems, including the xMAP system, are amenable to measuring several different markers in multiplex, and could be used to measure levels of antibodies at once. In some embodiments, other systems are used to assay a plurality of markers in multiplex.
  • profiling may be performed using any of the following systems: antigen microarrays, bead microarrays, nanobarcodes particle technology, arrayed proteins from cDNA expression libraries, protein in situ array, protein arrays of living transformants, universal protein array, lab-on-a-chip microfluidics, and peptides on pins.
  • Another type of clinical assay is a chemiluminescent assay to detect antibody binding.
  • assays including the VITROS Eci anti-HCV assay, antibodies are bound to a solid-phase support made up of microparticles in liquid suspension, and a surface fluorometer is used to quantify the enzymatic generation of a fluorescent product.
  • the biological sample shows the presence of S. pneumoniae (e.g., by detecting one or more polypeptide of Table 1 or 2 or an antibody that binds one of said polypeptides), one may administer a therapeutically effective amount of the compositions and therapies described herein to the patient.
  • the biological sample may comprise, for example, blood, semen, urine, vaginal fluid, mucus, saliva, feces, urine, cerebrospinal fluid, or a tissue sample.
  • the biological sample is an organ intended for transplantation.
  • the biological sample before the detection step, is subject to culture conditions that promote the growth of S. pneumoniae.
  • the diagnostic tests herein may be used to detect S. pneumoniae in a variety of samples, including samples taken from patients and samples obtained from other sources.
  • the diagnostic tests may be used to detect S. pneumoniae in food, drink, or ingredients for food and drink; on objects such as medical instruments, medical devices such as cochlear implants and pacemakers, shoes, clothing, furniture including hospital furniture, and drapes including hospital drapes; or in samples taken from the environment such as plant samples.
  • the tests herein may be performed on samples taken from animals such as agricultural animals (cows, pigs, chickens, goats, horses and the like), companion animals (dogs, cats, birds, and the like), or wild animals.
  • the tests herein may be performed on samples taken from cell cultures such as cultures of human cells that produce a therapeutic protein, cultures of bacteria intended to produce a useful biological molecule, or cultures of cells grown for research purposes.
  • This disclosure also provides a method of determining the location of a S. pneumoniae infection in a patient comprising: (a) administering a pharmaceutical composition comprising a labeled S. pneumoniae antibody or antigen-binding portion thereof to the patient, and (b) detecting the label, wherein binding indicates a S. pneumoniae infection in a particular location in the patient.
  • a diagnostic may also comprise comparing the levels of binding in the patient to a control.
  • the method further comprises, if the patient has a S. pneumoniae infection, treating the infection by administering a therapeutically effective amount of a S. pneumoniae -binding antibody or antigen-binding portion thereof to the patient.
  • the method further comprises, if the patient has a S.
  • the method may further comprise determining the location and/or volume of the S. pneumoniae in the patient. This method may be used to evaluate the spread of S. pneumoniae in the patient and determine whether a localized therapy is appropriate.
  • the anti- S. pneumoniae antibodies described herein may be used to make a prognosis of the course of infection.
  • the anti- S. pneumoniae antibodies herein may be detected in a sample taken from a patient. If antibodies are present at normal levels, it would indicate that the patient has raised an immune response against anti- S. pneumoniae . If antibodies are absent, or present at reduced levels, it would indicate that the patient is failing to raise a sufficient response against anti- S. pneumoniae , and a more aggressive treatment would be recommended.
  • antibodies present at reduced levels refers to antibodies that are present at less than 50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies typical in a patient with a normal immune system.
  • Antibodies may be detected by affinity for any of the antigens described herein (e.g., those in Table 1 and/or 2), for example using ELISA.
  • detection of specific S. pneumoniae antigens may be used to predict the progress and symptoms of S. pneumoniae infection in a patient. It will be understood by one of skill in the art that the methods herein are not limited to detection of S. pneumoniae .
  • Other embodiments include the detection of related bacteria including bacteria with proteins homologous to the proteins described in Table 1 or 2. Such related bacteria include, for example, other strains of Streptococcus.
  • the amount of antigen in each vaccine or immunogenic composition dose is selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses.
  • the dose is without significant, adverse side effects in typical vaccinees.
  • Such amount will vary depending upon which specific antigen is employed.
  • a dose will comprise 1-1000 ⁇ g of protein, in some instances 2-100 ⁇ g, for instance 4-40 ⁇ g.
  • the vaccine formulation comprises 1-1000 pig of the polypeptide and 1-250 ⁇ g of the adjuvant.
  • the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immunocompromised status) of a subject. When present, typically an adjuvant will be present in amounts from 1 ⁇ g-250 ⁇ g per dose, for example 50-150 ⁇ g, 75-125 ⁇ g or 100 ⁇ g.
  • only one dose of the vaccine is administered to achieve the results described above.
  • subjects receive one or more boost vaccinations, for a total of two, three, four or five vaccinations.
  • the number is three or fewer.
  • a boost vaccination may be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months. It may be advantageous to administer split doses of vaccines which may be administered by the same or different routes.
  • compositions described herein may take on a variety of dosage forms.
  • the composition is provided in solid or powdered (e.g., lyophilized) form; it also may be provided in solution form.
  • a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.
  • the composition will be administered in a dose escalation manner, such that successive administrations of the composition contain a higher concentration of composition than previous administrations. In some embodiments, the composition will be administered in a manner such that successive administrations of the composition contain a lower concentration of composition than previous administrations.
  • compositions are administered to a patient suffering from a disease in an amount sufficient to treat the patient.
  • Therapeutic applications of a composition described herein include reducing transmissibility, slowing disease progression, reducing bacterial viability or replication, or inhibiting the expression of proteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the levels at which they would occur in individuals who are not treated with the composition.
  • compositions are administered to a human or other mammal to induce an immune response that can inhibit the establishment of an infectious disease or other condition.
  • a composition may partially block the bacterium from establishing an infection.
  • the compositions are administered in combination with antibiotics.
  • antibiotics are used to treat pneumococcal infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and vancomycin.
  • the appropriate antibiotic may be selected based on the type and severity of the infection, as well as any known antibiotic resistance of the infection (Jacobs M R “Drug-resistant Streptococcus pneumoniae : rational antibiotic choices” Am J Med. 1999 May 3; 106(5A):19S-25S).
  • the vaccine formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled) or they can be administered by topical application.
  • the route of administration is intramuscular.
  • the route of administration is subcutaneous.
  • the route of administration is mucosal.
  • the route of administration is transdermal or intradermal
  • Certain routes of administration are particularly appropriate for vaccine formulations and immunogenic compositions comprising specified adjuvants.
  • transdermal administration is one suitable route of administration for S. pneumoniae vaccines comprising toxins (e.g. cholera toxin or labile toxin); in other embodiments, the administration is intranasal.
  • Vaccines formulated with Alphavirus replicons may be administered, for example, by the intramuscular or the subcutaneous route.
  • Vaccines comprising Monophosphory Lipid A (MPL), Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA) are suitable (inter alia) for intramuscular and subcutaneous administration.
  • a vaccine comprising resiquimod may be administered topically or subcutaneously, for example.
  • the vaccine formulation or immunogenic composition may be suitable for administration to a human patient, and vaccine or immunogenic composition preparation may conform to USFDA guidelines.
  • the vaccine formulation or immunogenic composition is suitable for administration to a non-human animal.
  • the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules.
  • LPS lipopolysaccharide
  • the vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects.
  • the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments.
  • the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water.
  • Other vaccine or immunogenic composition components may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation.
  • the pyrogens may be inactivated or destroyed prior to administration to a patient.
  • Raw materials for vaccines such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be sterile, and each lot of the vaccine may be tested for sterility.
  • the endotoxin levels in the vaccine fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non-intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water.
  • EU endotoxin
  • the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein.
  • the desired molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture).
  • the vaccine or immunogenic composition comprising purified subunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of protein from host cells in which the subunit proteins were expressed, relative to the amount of purified subunit.
  • the desired polypeptides are substantially free of nucleic acids and/or carbohydrates.
  • the vaccine or immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or RNA.
  • At least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type are present in the preparation (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • the vaccine or immunogenic composition has low or no toxicity, within a reasonable risk-benefit ratio.
  • the vaccine or immunogenic composition comprises ingredients at concentrations that are less than LD 50 measurements for the animal being vaccinated. LD 50 measurements may be obtained in mice or other experimental model systems, and extrapolated to humans and other animals. Methods for estimating the LD 50 of compounds in humans and other animals are well-known in the art.
  • a vaccine formulation or immunogenic composition might have an LD 50 value in rats of greater than 100 g/kg, greater than 50 g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg.
  • a vaccine formulation or immunogenic composition that comprises a toxin such as botulinum toxin (which can be used as an adjuvant) should contain significantly less than the LD 50 of botulinum toxin.
  • formulations suitable for introduction of the vaccine formulations or pharmaceutical composition vary according to route of administration.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the cells can be administered intravenously or parenterally.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
  • the antigens can be made into aerosol formulations (e.g., they can be “nebulized”) to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
  • Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptides or packaged nucleic acids with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the polypeptides or packaged nucleic acids with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • S. pneumoniae vaccines and immunogenic compositions described herein may be produced using a variety of techniques.
  • a polypeptide may be produced using recombinant DNA technology in a suitable host cell.
  • a suitable host cell may be bacterial, yeast, mammalian, or other type of cell.
  • the host cell may be modified to express an exogenous copy of one of the relevant polypeptide genes.
  • the gene is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence.
  • the promoter is inducible or repressible.
  • the gene may be present on an extrachromosomal plasmid, or may be integrated into the host genome.
  • One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and may be desirable.
  • the nucleic acid may be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same.
  • the gene is codon-optimized to improve expression in a particular host.
  • the nucleic acid may be produced, for example, by PCR or by chemical synthesis.
  • a polypeptide may be isolated from it.
  • the isolation may be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).
  • a method of manufacture comprising mixing one or more polypeptides or an immunogenic fragment or variant thereof with a carrier and/or an adjuvant.
  • antigens for inclusion the vaccine formulations and immunogenic compositions may be produced in cell culture.
  • One method comprises providing one or more expression vectors and cloning nucleotides encoding one or more polypeptides selected from polypeptides having an amino acid sequence of Table 1 or Table 2, then expressing and isolating the polypeptides.
  • the immunogenic polypeptides described herein, and nucleic acid compositions that express the polypeptides can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal.
  • packs or dispenser devices that contain one or more unit dosage forms are provided.
  • instructions for administration of the compounds will be provided with the packaging, along with a suitable indication on the label that the compound is suitable for treatment of an indicated condition, such as those disclosed herein.
  • Each open reading frame predicted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major histocompatibility complex (MHC). Each construct was then expressed in E. coli , and full-length expression validated by a surrogate assay that identifies the tag in the context of MHC. The screen is described in more detail in International Application WO 2010/002993.
  • the library was pooled such that four induced library clones were present in each well.
  • an aliquot of the pooled library was added to peritoneal-derived macrophages.
  • the macrophages were allowed to bind the tagged S. pneumoniae antigens via the MHC. After 2 hr at 37° C., the macrophages were washed with PBS. The macrophages were then fixed with 1% paraformaldehyde for 15 min and washed extensively with PBS. 10 5 T cells were added to each well in 200 ⁇ L of RP-10 media. The T cells had previously been isolated from mice that had been immunized 2 times with killed S. pneumoniae bacteria with cholera toxin adjuvant. The assay plates were incubated for 72 hrs at 37° C. The amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay. The threshold for a positive result was set at two standard deviations above the mean of all samples.
  • a secondary screen was used to determine which antigen(s) out of the four clones in each well induced the positive response observed in the pooled screen described in Example 1. All the clones in each positive pool were pulsed individually onto peritoneal macrophages in duplicate wells. T cells isolated from immunized mice from the same genetic background as the initial screen were used to screen the pulsed macrophages using the IL-17 assay described in Example 1. Individual antigens that induced an average response in the duplicate wells greater than two standard deviations above the mean of negative control samples were considered positive responses. The library plasmids present in these positive clones were sequenced to confirm the identity of the antigen. The antigens SP_1574, SP_1655, SP_2106, SP_0148.
  • SP_2002, SP_0612, SP_1988, SP_0484, SP_0847, SP_1527, SP_0542, SP_0441, SP_0350, SP_0014, SP_1965, SP_0117, and SP_2108 were confirmed using this method.
  • CD4+ T cells and CD14+ monocytes were isolated from peripheral blood acquired from human donors.
  • the monocytes were differentiated into dendritic cells by culturing them in GM-CSF and IL-4 containing media, essentially as described in Tedder T F and Jansen P J (1997 “Isolation and generation of human dendritic cells.” Current Protocols in Immunology Supp 23: 7.32.1-7.32.16). After five days in culture, the dendritic cells were seeded into 384 well plates.
  • the CD4+ T cells were expanded in culture to ensure sufficient quantities.
  • Each open reading frame predicted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major histocompatibility complex (MHC). Each construct was then expressed in E. coli , and full-length expression validated by a surrogate assay that identifies the tag in the context of MHC.
  • MHC major histocompatibility complex
  • the library was pooled such that four induced library clones were present in each well.
  • an aliquot of the pooled library was added to the seeded dendritic cells in 384-well plates.
  • the dendritic cells were fixed with 1% paraformaldehyde for 15 min and washed extensively with phosphate buffer and lysine buffer.
  • 40,000 of the CD4+ T cells in 70 ⁇ L of RP-10 media were added to each well of a 384-well plate.
  • the assay plates were incubated for 3 days at 37° C.
  • the amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay.
  • the threshold for a positive result was set at two standard deviations above the mean of all samples, two standard deviations above the mean of negative controls, or 1.78 times the median absolution deviation of the data set. Positive pools were then deconvoluted as described in Example 4.
  • deconvolution was performed by comparing the results of two pool screens.
  • two different sets of pools were prepared, so that a polypeptide was with three different polypeptides between the first and second pools. Consequently, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in positive pools in both the first and second sets.
  • a pool was identified as positive if it was at least 1.78 times the median absolution deviation of the data set.
  • An antigen was identified as a positive hit if it was positive in at least two repeated secondary screens.
  • the antigens SP2108, SP0641, SP1393, SP0024, SP0641.1, SP1072, SP1384 and SP2032 were identified using the above approach.
  • the SP2108 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 7) and SP1634 polypeptide were formulated as vaccine compositions using 4 ⁇ g of the polypeptide in combination with 1 ⁇ g cholera toxin. For combinations, 4 ⁇ g of each polypeptide was used.
  • the compositions were administered intranasally to C57BU/6 mice three times, one week apart. The subjects were then allowed to rest for 3 weeks, and bled at that time for immunogenicity. For this assay, heparinized whole blood was collected from the retrograde orbital sinus. The total PBMC were stimulated with either killed whole cells (WCC) or a combination of the three polypeptides in round bottomed tubes for three days.
  • WCC killed whole cells
  • results are shown in FIGS. 1 and 2 .
  • the subjects were allowed to rest an additional 2 weeks, at which time they were challenged with intranasal administration of S. pneumoniae .
  • the subjects were sacrificed a week later, and the number of colony-forming units (CFU) was counted from nasal washes. Results are shown in FIG. 3 .
  • CFU colony-forming units
  • Example 5 This example used the same protocols as Example 5, except that only two doses of the vaccine composition were administered.
  • the SP0882 polypeptide (SEQ ID NO: 2) and SP0314 polypeptides (see Table 2) were used in conjunction with the three polypeptides tested in Example 5.
  • Results of the immunogenicity assay are shown in FIGS. 4 and 5 .
  • Results of the colonization assay are shown in FIG. 6 .
  • This example used a protocol similar to that of Example 5, except that two doses of the vaccine composition were administered, one week apart.
  • the mice were challenged intranasally with live type 6B S. pneumoniae .
  • One week later the bacterial burden was assessed in each mouse by plating a nasal lavage on selective media and counting CFU.
  • mice were immunized, challenged with S. pneumoniae , and the number of CFU was recorded. The results of this experiment are shown in FIG. 8 .

Abstract

Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. The present disclosure provides, inter alia, certain highly effective vaccines and pharmaceutical compositions in Streptococcus pneumoniae. The antigens may be used therapeutically or prophylactically.

Description

RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 12/826,084, filed on Jun. 29, 2010, now U.S. patent Ser. No. 10/105,412, which claims the benefit of the filing date of U.S. Provisional Application No. 61/221,541, filed Jun. 29, 2009, U.S. Provisional Application No. 61/240,616, filed Sep. 8, 2009, U.S. Provisional Application No. 61/240,598, filed Sep. 8, 2009, and U.S. Provisional Application No. 61/316,267, filed Mar. 22, 2010. The entire teachings of the referenced applications are expressly incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFSWeb and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 13, 2019, is named 2007781-0200 SL.txt and is 142932 bytes in size.
I. BACKGROUND
Pneumococcal disease continues to be a leading cause of sickness and death in the United States and throughout the world. Each year, millions of cases of pneumonia, meningitis, bacteremia, and otitis media are attributed to infection with the pathogen Streptococcus pneumoniae. S. pneumoniae is a Gram-positive encapsulated coccus that colonizes the nasopharynx in about 5-10% of healthy adults and 20-40% of healthy children. Normal colonization becomes infectious when S. pneumoniae is carried into the Eustachian tubes, nasal sinuses, lungs, bloodstream, meninges, joint spaces, bones and peritoneal cavity. S. pneumoniae has several virulence factors that enable the organism to evade the immune system. Examples include a polysaccharide capsule that prevents phagocytosis by host immune cells, proteases that inhibit complement-mediated opsonization, and proteins that cause lysis of host cells. In the polysaccharide capsule, the presence of complex polysaccharides forms the basis for dividing pneumococci into different serotypes. To date, 93 serotypes of S. pneumoniae have been identified.
Various pharmaceutical compositions have been used to harness an immune response against infection by S. pneumoniae. A polyvalent pneumococcal vaccine, PPV-23, was developed for preventing pneumonia and other invasive diseases due to S. pneumoniae in the adult and aging populations. The vaccine contains capsular polysaccharides (CPs) from 23 serotypes of S. pneumoniae. As T cell independent antigens, these CPs induce only short-lived antibody responses, necessitating repeated doses, which increases the risk of immunological tolerance. The antibodies raised against S. pneumoniae, termed anticapsular antibodies, are recognized as protective in adult and immunocompetent individuals. However, children under 2 years of age and immunocompromised individuals, including the elderly, do not respond well to T-cell independent antigens and, therefore, are not afforded optimal protection by PPV-23. A second S. pneumoniae vaccine, Prevnar, includes bacterial polysaccharides from 7 S. pneumoniae strains conjugated to the diphtheria toxoid protein. This vaccine induces both B and T cell responses. However, because it only protects against 7 pneumococcal serotypes, serotype replacement can render Prevnar ineffective. PPV-23 suffers from the same limitation. Serotype replacement has already been demonstrated in several clinical trials and epidemiologic studies, and raises the possibility that different formulations of the vaccines will need to be developed, presumably at even higher cost. Furthermore, both PPV-23 and Prevnar are expensive to manufacture, greatly limiting their availability in the developing world.
Thus, there remains a need to design more effective pharmaceutical compositions than the current strategies offer. In particular, such compositions need to incorporate novel or specific antigens that elicit an immune response against S. pneumoniae.
II. SUMMARY
Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. While DNA and protein sequence information for S. pneumoniae has been known for some time, and researchers have long attempted to produce vaccines against S. pneumoniae, a major problem was how to identify protective polypeptides from among the approximately 2100 genes in the S. pneumoniae genome. The instant application presents the results of whole-genome screens designed to identify the most immunogenic proteins in the S. pneumoniae genome. Several of the hits from the screen have been shown to protect against S. pneumoniae colonization in a mouse model. Accordingly, the present disclosure provides, inter alia, certain highly effective vaccines in Streptococcus pneumoniae. The vaccines may be used therapeutically or prophylactically. The present disclosure also provides specific antigens and methods for using the antigens to elicit an immune response against S. pneumoniae.
The present disclosure provides, for example, a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence consisting of SEQ ID NO: 11 or an immunogenic fragment thereof. In addition, the present disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence comprising SEQ ID NO: 12.
Furthermore, the instant application provides a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof.
This application provides, inter alia, a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein.
The present disclosure further provides an immunogenic composition comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid sequences comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof.
III. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with killed, unencapsulated whole cell S. pneumoniae, as described in Example 5. The left panel shows the data in scatter format, and the right panel shows the average and standard deviation for each sample. Immunization group “All 3” represents animals immunized with a combination of SP2108, SP0148, and SP1634.
FIG. 2 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with a combination of three proteins (SP2108, SP0148, and SP1634), as described in Example 5.
FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae, as described in Example 5. 003 represents a control irrelevant antigen.
FIG. 4 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated with killed, unencapsulated whole cell S. pneumoniae, as described in Example 6.
FIG. 5 shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin, then stimulated by the indicated proteins and combination, as described in Example 5.
FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae, as described in Example 6. The HSV-2 protein ICP47 with the gene name US12 (NP_044543.1, NC_001798.1; shown in the figure as 003) and ovalbumin (OVA) represent control antigens.
FIG. 7 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae, as described in Example 7.
FIG. 8 shows the number of S. pneumoniae colonies obtained from a nasal wash in BALB/c mice that were immunized with the indicated protein(s) and cholera toxin, then challenged with intranasal administration of S. pneumoniae, as described in Example 8.
 IV. DETAILED DESCRIPTION
A. Specific Polypeptides and Nucleic Acids for Use in S. pneumoniae Vaccines and Immunogenic Compositions
This application describes S. pneumoniae vaccines that include one or more of the polypeptides or genes listed in Table 1, or variants or fragments thereof as described below. The vaccine may include a polypeptide that comprises a sequence of Table 1 or a variant or immunogenic fragment thereof or a polypeptide that consists of a sequence of Table 1 or a variant or immunogenic fragment thereof. The DNA and protein sequence of each gene and polypeptide may be found by searching for the Locus Tag in the publicly available database, Entrez Gene (on the NCBI NIH web site on the World Wide Web, at www.ncbi.nlm.nih.gov/sites/entrez?db=gene), in the Streptococcus pneumoniae TIGR4 genome, and the indicated sequences are also included in this application.
TABLE 1
Immunogenic polypeptides for vaccine formulations
Protein DNA DNA GenBank
Locus tag name and SEQ ID SEQ ID Accession No.
description No. No. (from Mar. 30, 2010)
SP0024 1 — NC_003028.3|:27381-
27878
SP0882 2 — NC_003028.3|:831.804-
832628
SP0882N 3 24 —
SP0882 with exogenous 4 25 —
leader
SP0882N with exogenous 5 26 —
leader
SP0148 lacking signal 6 27 —
sequence
SP0148 including signal 7 28 —
sequence
SP1072
8 — NC_003028.3|:1008420-
1010180
SP2108 including signal 9 — NC_003028.3|:2020750-
sequence 2022021
SP2108 lacking signal 10 29 —
sequence
SP0641M 11 30 —
SP0641 12 — NC_003028.3|:2020750-
2022021
SP0641N 13 31 —
SP0882 consensus 14 — —
SP0882N consensus 15 — —
SP0882 consensus with 16 — —
exogenous leader
SP0882N consensus with 17 — —
exogenous leader
SP0148 consensus lacking 18 — —
signal sequence
SP0148 consensus 19 — —
including signal sequence
SP2108 consensus 20 — —
lacking signal sequence
SP2108 consensus 21 — —
including signal sequence
SP1634 22 — NC_003028.3|:1534348-
1535421
SP0314 23 — NC_003028.3|:287483-
290683
Certain polypeptides of Table 1, and variants thereof, are described in greater detail below.
1. SP0024 (SEQ ID NO: 1) and Variants Thereof
SP0024 represents a hypothetical protein of 165 amino acids, containing a conserved carbonic anhydrase domain that extends from amino acid 27 to amino acid 163. Based on this consensus motif, SP0024 may be a zinc-binding protein.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0024. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 150, 125, or 100 consecutive amino acids from SP0024.
2. SP0882 (SEQ ID NO: 2) and Variants Thereof
SP0882 is a conserved hypothetical protein of 274 amino acids. Much of the protein (amino acids 2-270) forms an esterase or lipase-like region.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0882. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids from SP0882.
One particular truncation variant named SP0882N consists of the N-terminal 130 amino acids of SP0882, and is shown as SEQ ID NO: 3. SP0882N includes a region that is particularly well conserved among different serotypes. In certain embodiments, a polypeptide comprising SP0882 or SP0882N, or an immunogenic fragment of either, also comprises an exogenous leader sequence. The leader sequence may be, for example, the leader sequence of SP2108. Two exemplary such polypeptides are SEQ ID NOS: 4 and 5.
Variants of DNA and protein sequences of SP0882 are described, inter alia, in US Patent Application Publication No. 2009/0215149 and International Applications WO2002/077021, WO98/18931, and WO2007/106407. A variant of SP0882N is disclosed in International Application WO2008/146164.
Sequence variation occurs at the protein level between different S. pneumoniae serotypes, and consensus sequences illustrating combinations of SP0882 sequences from different S. pneumoniae serotypes are provided as SEQ ID NOS: 14-17. Accordingly, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-17, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 2-5).
Nucleic acid sequences encoding different variants of SP0882 are provided as SEQ ID NOS: 24-26, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) could encode these polypeptides.
3. SP0148 (SEQ ID NO: 7) and Variants Thereof
The protein SP0148 is named “ABC transporter, substrate-binding protein”. Proteins of this class are typically extracellular proteins that interact transiently with a transmembrane protein complex. Such complexes use energy generated by ATP hydrolysis to translocate specific substrates across a cell membrane. SP0148 is a 276 amino acid protein that contains a conserved PBPb (periplasmic binding protein) domain, spanning amino acids 40-246, which is typical of membrane-bound transport complexes. In addition. SB0148 has a bacterial extracellular solute-binding proteins, family 3 domain which is largely co-extensive with the PBPb domain and extends from amino acid 40 to 244. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP0148 comprising or lacking one or more of said domains and motifs.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0148. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids from SP0148.
Endogenous SP0148 comprises a putative signal sequence that may direct its secretion. In some embodiments, a variant of SP0148 that lacks the signal sequence (SEQ ID NO: 6) is used. The polypeptide of SEQ ID NO: 6 is encoded by the nucleic acid of SEQ ID NO: 27, although other nucleic acid sequences (including codon-optimized sequences) may be used. SEQ ID NO: 28 encodes the full length sequence of SP0148 used in the screens herein.
Variants of the amino acid sequence and nucleotide sequence of SP0148 may be found in U.S. Patent Application Publication No. 2005/0020813. U.S. Pat. Nos. 7,378,514 and 7,504,110, and European Patent Application No. EP1572868 and EP1855717.
Consensus sequences illustrating combinations of SP0148 sequences from different S. pneumoniae serotypes are provided as SEQ ID NOS: 18 and 19. Accordingly, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 18-19, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 6 or 7).
4. SP1072 (SEQ ID NO: 8) and Variants Thereof
SP1072, also known as dnaG, is a DNA primase enzyme that catalyzes formation of an RNA primer which allows DNA polymerase to initiate DNA replication. A protein of 586 amino acids, SP1072 contains several conserved motifs. Beginning at the N-terminus, amino acids 2-96 form a zinc finger domain, the DNA primase catalytic core spans amino acids 122-250, and a highly conserved topoisomerase-primase (TORPIM) nucleotidyl transferase/hydrolase domain region extends from amino acid 258 to 330. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP1072 comprising or lacking one or more of said domains and motifs.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected form SP1072. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP1072.
5. SP2108 (SEQ ID NO: 9) and Variants Thereof
The polypeptide SP2108 is 423 amino acids in length and is alternatively known as MalX, maltose/maltodextrin ABC transporter, or maltose/maltodextrin-binding protein. Much of the protein (amino acids 3-423) is classified as a MalE (Maltose-binding periplasmic) domain. In addition. SP2108 contains a signal sequence that directs its secretion. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP2108 comprising one or more of said domains and motifs.
In some embodiments, the compositions and methods herein call for the use of an SP2108 variant that lacks the signal sequence. This variant is represented by polypeptide sequence SEQ ID NO: 10 and may be encoded by, for example, a nucleic acid according to SEQ ID NO: 29.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP2108. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP2108.
Consensus sequences illustrating combinations of SP2108 sequences from different serotypes are provided as SEQ ID NOS: 20 and 21. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 20-21, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 9 or 10).
6. SP0641 (SEQ ID NO: 12) and Variants Thereof
At 2144 amino acids in length, SP0641 is also known as PrtA, a cell wall-associated serine protease. Full-length SP0641 contains a number of conserved motifs: the PA_2 motif, extending between amino acids 485 and 597, which may form a protein binding surface; the Fn3-like domain (amino acids 800-939); and two predicted catalytic domains of the SS8 C5a type located at amino acids 226-449 and 639-777. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP0641 comprising or lacking one or more of said domains and motifs.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0641. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 consecutive amino acids from SP0641.
Certain other truncation mutants of SP0641 may also be used. For instance, the polypeptide designated SP0641N (SEQ ID NO: 13) consists of 661 amino acids corresponding to amino acids 24-684 near the N-terminus of SP0641. Roughly adjacent to SP0641N (and corresponding to amino acids 686-1333 of SP0641) lies the 648 residue region captured by the truncation variant SP0641M (SEQ ID NO: 11).
Variants of SP0641 are disclosed in, for example, U.S. Pat. Nos. 7,338,786, 6,573,082, and 7,132,107, as well as International Application WO00/06738.
SEQ ID NOS: 30 and 31 display the DNA sequences of SP0641M and SP0641N, respectively, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) could encode SP0641.
Polypeptides homologous to the polypeptides of Tables 1 and 2 (for example, SP0024, 0882, 0882N, 0148 with or without a signal sequence, 1072, SP1028 with or without a signal sequence, SP0641, SP0641M, or SP0641N) may also be used in the compositions and methods disclosed herein. Individual strains of S. pneumoniae contain numerous mutations relative to each other, and some of these result different protein sequences between the different strains. One of skill in the art may readily substitute an amino acid sequence, or a portion thereof, with the homologous amino acid sequence from a different S. pneumoniae strain. In certain aspects, this application provides immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the polypeptides of Tables 1 and 2 or an immunogenic fragment thereof. Serotypic variation may be used to design such variants of the polypeptides of Tables 1 and 2.
In some embodiments, the vaccine compositions herein comprise a fragment of a protein of Table 1 or 2 (for example, fragments of SP0024, SP0882, SP0882N, 0SP148 with or without a signal sequence, SP1072, SP1028 with or without a signal sequence, SP0641, SP0641M, or SP0641N). In some embodiments, this application provides truncation mutants that are close in size to the polypeptide of Table 1 or 2 (for example, one of SEQ ID NOS: 1-13). For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from one or both termini. Internal deletions, e.g., of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.
In certain embodiments the vaccine formulation comprises one or more polypeptides having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-21. In certain embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 14-21 wherein from 1-5, 1-10, or 1-20 amino acid residues are removed from the N-terminus, C-terminus, or both. In certain embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 14-21 wherein from 1-10 amino acid residues are removed from the N-terminus, C-terminus, or both. For instance, 10 amino acid residues may be removed from each of the N-terminus and C-terminus resulting in a protein with 20 amino acid residues removed.
In addition to those nucleic acids and polypeptides described in Table 1 above, this application also provides immunogenic compositions that include one or more of the polypeptides or genes listed in Table 1 and/or Table 2, or variants or fragments thereof as described herein. The DNA and protein sequence of each gene and protein may be found by searching for the Locus Tag in the publicly available database, Entrez Gene, as described above.
TABLE 2
Immunogenic proteins identified in human and mouse screens
Locus tag Protein accession DNA accession number (from
name number Mar. 30, 2010)
SP1574 AAK75660.1 NC_003028.3|:c1481367-1480609
SP1655 AAK75734.1 NC_003028.3|:c1557922-1557230
SP2106 AAK76165.1 NC_003028.3|:c2018657-2016399
SP1473 AAK75567.1 NC_003028.3|:c1386534-1386277
SP0605 AAK74757.1 NC_003028.3|:571604-572485
SP1177 AAK75286.1 NC_003028.3|:c1115580-1115317
SP0335 AAK74510.1 NC_003028.3|:306559-306876
SP0906 AAK75031.1 NC_003028.3|:c859160-859029
SP1828 AAK75901.1 NC_003028.3|:c1740010-1739000
SP2157 AAK76211.1 NC_003028.3|:c2072146-2070995
SP1229 AAK75335.1 NC_003028.3|:c1163388-1161718
SP1128 AAK75238.1 NC_003028.3|:1061773-1063077
SP1836 AAK75909.1 NC_003028.3|:1746104-1746280
SP1865 AAK75937.1 NC_003028.3|:c1772987-1771923
SP0904 AAK75029.1 NC_003028.3|:c858126-857311
SP0765 AAK74903.1 NC_003028.3|:724170-725207
SP1634 AAK75714.1 NC_003028.3|:1534348-1535421
SP0418 AAK74581.1 NC_003028.3|:396692-396916
SP1923 AAK75991.1 NC_003028.3|:c1833311-1831896
SP1313 AAK75991.1 NC_003028.3|:c1833311-1831896
SP0775 AAK74913.1 NC_003028.3|:731798-732070
SP0314 AAK74491.1 NC_003028.3|:287483-290683
SP0912 AAK75037.1 NC_003028.3|:864707-865465
SP0159 AAK74341.1 NC_003028.3|:c157554-156292
SP0910 AAK75035.1 NC_003028.3|:863462-863734
SP2148 AAK76205.1 NC_003028.3|:2062144-2063373
SP1412 AAK75510.1 NC_003028.3|:c1332393-1331605
SP0372 AAK74539.1 NC_003028.3|:350268-350597
SP1304 AAK75407.1 NC_003028.3|:c1232491-1232390
SP2002 AAK76069.1 NC_003028.3|:c1906183-1905446
SP0612 AAK74764.1 NC_003028.3|:579708-579806
SP1988 AAK76055.1 NC_003028.3|:c1892598-1890565
SP0484 AAK74643.1 NC_003028.3|:465572-466402
SP0847 AAK74978.1 NC_003028.3|:794144-795202
SP1527 AAK75616.1 NC_003028.3|:c1439494-1437536
SP0542 AAK74699.1 NC_003028.3|:515940-516059
SP0441 AAK74602.1 NC_003028.3|:414869-415057
SP0350 AAK74523.1 NC_003028.3|:323990-324625
SP0014 AAK74207.1 NC_003028.3|:14450-14929
SP1965 AAK76032.1 NC_003028.3|:c1873279-1873073
SP0117 AAK74303.1 NC_003028.3|:118423-120657
SP0981 AAK75102.1 NC_003028.3|:927115-928056
SP2229 AAK76277.1 NC_003028.3|:c2148627-2147602
SP2136 AAK76194.1 NC_003028.3|:c2048521-2046656
SP1179 AAK75288.1 NC_003028.3|:1116230-1118389
SP1174 AAK75283.1 NC_003028.3|:c1110717-1108258
SP2216 AAK76264.1 NC_003028.3|:c2136445-2135267
SP1393 AAK75491.1 NC_003028.3|:1316756-1318027
SP0641.1 Amino acids 28- Nucleotides 603976-606910 of
1006 of NC_003028.3
AAK74791.1
(which is full-
length SP0641)
SP1384 AAK75482.1 NC_003028.3|:c1309464-1308967
SP2032 AAK76097.1 NC_003028.3|:c1939994-1938321
Typically, the polypeptides present in compounds of the invention are immunogenic, either alone or as a variant, which includes polypeptides fused to another polypeptide or mixed with or complexed to an adjuvant. Variants also include sequences with less than 100% sequence identity, as described herein. In certain embodiments, an antigen of Table 1 or 2 is provided as a full length polypeptide. In addition, one may use fragments, precursors and analogs that have an appropriate immunogenicity.
These polypeptides may be immunogenic in mammals, for example mice, guinea pigs, or humans. An immunogenic polypeptide is typically one capable of raising a significant immune response in an assay or in a subject. For instance, an immunogenic polypeptide may increase the amount of IL-17 produced by T cells. The IL-17 assay described in Examples 1-4 is an example of an assay that may be used to identify an immunogenic polypeptide. Alternatively, an immunogenic polypeptide may (i) induce production of antibodies, e.g., neutralizing antibodies, that bind to the polypeptide (ii) induce T H1 immunity. (iii) activate the CD8+ CTL response, for example by increasing CD8+ T cells and/or increasing localization of CD8+ T cells to the site of infection or reinfection, (iv) induce T H17 immunity, and/or (v) activate innate immunity. In some embodiments, an immunogenic polypeptide causes the production of a detectable amount of antibody specific to that antigen.
In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%, 60% or 70% identity to human autoantigens and/or gut commensal bacteria (e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia and Lactobacillus species). Examples of human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.
The present disclosure provides, for example, a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof. In certain embodiments, the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof. Here, the term “different” signifies that each of said two peptides originates from a different sequence selected from SEQ ID NOS: 1-13.
The vaccine formulation may also comprise one or more polypeptides having an amino acid sequence consisting of any of SEQ ID NOS: 1-11.
In some embodiments, the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragment thereof. (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or an immunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 or an immunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13 or an immunogenic fragment thereof. Examples of such combinations are listed below:
SEQ ID NO: 1 and SEQ ID NO: 2
SEQ ID NO: 1 and SEQ ID NO: 3
SEQ ID NO: 1 and SEQ ID NO: 4
SEQ ID NO: 1 and SEQ ID NO: 5
SEQ ID NO: 1 and SEQ ID NO: 6
SEQ ID NO: 1 and SEQ ID NO: 7
SEQ ID NO: 1 and SEQ ID NO: 8
SEQ ID NO: 1 and SEQ ID NO: 9
SEQ ID NO: 1 and SEQ ID NO: 10
SEQ ID NO: 1 and SEQ ID NO: 11
SEQ ID NO: 1 and SEQ ID NO: 12
SEQ ID NO: 1 and SEQ ID NO: 13
SEQ ID NO: 2 and SEQ ID NO: 6
SEQ ID NO: 2 and SEQ ID NO: 7
SEQ ID NO: 2 and SEQ ID NO: 8
SEQ ID NO: 2 and SEQ ID NO: 9
SEQ ID NO: 2 and SEQ ID NO: 10
SEQ ID NO: 2 and SEQ ID NO: 11
SEQ ID NO: 2 and SEQ ID NO: 12
SEQ ID NO: 2 and SEQ ID NO: 13
SEQ ID NO: 3 and SEQ ID NO: 6
SEQ ID NO: 3 and SEQ ID NO: 7
SEQ ID NO: 3 and SEQ ID NO: 8
SEQ ID NO: 3 and SEQ ID NO: 9
SEQ ID NO: 3 and SEQ ID NO: 10
SEQ ID NO: 3 and SEQ ID NO: 11
SEQ ID NO: 3 and SEQ ID NO: 12
SEQ ID NO: 3 and SEQ ID NO: 13
SEQ ID NO: 4 and SEQ ID NO: 6
SEQ ID NO: 4 and SEQ ID NO: 7
SEQ ID NO: 4 and SEQ ID NO: 8
SEQ ID NO: 4 and SEQ ID NO: 9
SEQ ID NO: 4 and SEQ ID NO: 10
SEQ ID NO: 4 and SEQ ID NO: 11
SEQ ID NO: 4 and SEQ ID NO: 12
SEQ ID NO: 4 and SEQ ID NO: 13
SEQ ID NO: 5 and SEQ ID NO: 6
SEQ ID NO: 5 and SEQ ID NO: 7
SEQ ID NO: 5 and SEQ ID NO: 8
SEQ ID NO: 5 and SEQ ID NO: 9
SEQ ID NO: 5 and SEQ ID NO: 10
SEQ ID NO: 5 and SEQ ID NO: 11
SEQ ID NO: 5 and SEQ ID NO: 12
SEQ ID NO: 5 and SEQ ID NO: 13
SEQ ID NO: 6 and SEQ ID NO: 8
SEQ ID NO: 6 and SEQ ID NO: 9
SEQ ID NO: 6 and SEQ ID NO: 10
SEQ ID NO: 6 and SEQ ID NO: 11
SEQ ID NO: 6 and SEQ ID NO: 12
SEQ ID NO: 6 and SEQ ID NO: 13
SEQ ID NO: 7 and SEQ ID NO: 8
SEQ ID NO: 7 and SEQ ID NO: 9
SEQ ID NO: 7 and SEQ ID NO: 10
SEQ ID NO: 7 and SEQ ID NO: 11
SEQ ID NO: 7 and SEQ ID NO: 12
SEQ ID NO: 7 and SEQ ID NO: 13
SEQ ID NO: 8 and SEQ ID NO: 9
SEQ ID NO: 8 and SEQ ID NO: 10
SEQ ID NO: 8 and SEQ ID NO: 11
SEQ ID NO: 8 and SEQ ID NO: 12
SEQ ID NO: 8 and SEQ ID NO: 13
SEQ ID NO: 9 and SEQ ID NO: 11
SEQ ID NO: 9 and SEQ ID NO: 12
SEQ ID NO: 9 and SEQ ID NO: 13
SEQ ID NO: 10 and SEQ ID NO: 11
SEQ ID NO: 10 and SEQ ID NO: 12
SEQ ID NO: 10 and SEQ ID NO: 13
In certain embodiments, the vaccine formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising one of SEQ ID NOS: 1-10. In certain such embodiments, the vaccine formulation comprises at least three polypeptides, each polypeptide belonging to a different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragment thereof. (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or an immunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 or an immunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13 or an immunogenic fragment thereof. Examples of such combinations are listed below:
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 7
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 6
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 7
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 6
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 7
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 8
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 8
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 9
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 10
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 11
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 12
SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 13
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 8
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 9
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 10
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 11
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 12
SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 13
SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 8
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 9
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 10
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 11
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 12
SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 13
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 8
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 9
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 10
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 11
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 12
SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 13
SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 8
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 9
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 10
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 11
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 12
SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 13
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 8
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 9
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 10
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 11
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 12
SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 13
SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 8
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 9
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 10
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 11
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 12
SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 13
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 8
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 9
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 10
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 11
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 12
SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 13
SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 9
SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 10
SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 11
SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 12
SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 13
SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 13
SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 11
SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 12
SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 13
SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 11
SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 12
SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 13
In some embodiments, the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof. In certain such embodiments, the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i)-(iii): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragment thereof. Examples of such combinations are listed below:
SEQ ID NO: 14 and SEQ ID NO: 18
SEQ ID NO: 14 and SEQ ID NO: 19
SEQ ID NO: 14 and SEQ ID NO: 20
SEQ ID NO: 14 and SEQ ID NO: 21
SEQ ID NO: 15 and SEQ ID NO: 18
SEQ ID NO: 15 and SEQ ID NO: 19
SEQ ID NO: 15 and SEQ ID NO: 20
SEQ ID NO: 15 and SEQ ID NO: 21
SEQ ID NO: 16 and SEQ ID NO: 18
SEQ ID NO: 16 and SEQ ID NO: 19
SEQ ID NO: 16 and SEQ ID NO: 20
SEQ ID NO: 16 and SEQ ID NO: 21
SEQ ID NO: 17 and SEQ ID NO: 18
SEQ ID NO: 17 and SEQ ID NO: 19
SEQ ID NO: 17 and SEQ ID NO: 20
SEQ ID NO: 17 and SEQ ID NO: 21
SEQ ID NO: 18 and SEQ ID NO: 20
SEQ ID NO: 18 and SEQ ID NO: 21
SEQ ID NO: 19 and SEQ ID NO: 20
SEQ ID NO: 19 and SEQ ID NO: 21
In some aspects, a vaccine formulation comprising one or more of SEQ ID NOS: 14-21 further comprises a polypeptide having an amino acid sequence comprising any of SEQ ID NOS: 1-13.
In certain embodiments, the vaccine formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21 or an immunogenic fragment thereof. In certain such embodiments, the vaccine formulation comprises three of (i)-(iii): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragment thereof. Examples of such combinations are listed below:
SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 20
SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 21
SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 20
SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 21
SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 20
SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21
SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20
SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 21
SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20
SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 21
SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20
SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21
SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 20
SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 21
SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 20
SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21
A polypeptide may comprise one or more immunogenic portions and one or more non-immunogenic portions. The immunogenic portions may be identified by various methods, including protein microarrays, ELISPOT/ELISA techniques, and/or specific assays on different deletion mutants (e.g., fragments) of the polypeptide in question. Immunogenic portions may also be identified by computer algorithms. Some such algorithms, like EpiMatrix (produced by EpiVax), use a computational matrix approach. Other computational tools for identifying antigenic epitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted by Dana Farber Cancer Institute on the world wide web at immunax.dfci.harvard.edu/PEPVAC), and MHCPred (which uses a partial least squares approach and is hosted by The Jenner Institute on the world wide web at www.jenner.ac.uk/MHCPred). An immunogenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by algorithm. Peptides identified by the tools described above include the following:
SP2108 SP0148 SP1634 SP0882 SP0314
Fragments Fragments Fragments Fragments Fragments
(SEQ ID NOS (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS
34-57, 58-82, 83-109, 110-130, 131-169,
respectively, respectively, respectively, respectively, respectively,
in order of in order of in order of in order of in order of
appearance) appearance) appearance) appearance) appearance)
AIIDGPWKA ALGLVAAGV RLLDLAPQV HLDNLVLKV MLKDKIAFL
VMMAPYDRV ELTGYEIEV MLEIPAHQI DLIAGRVHL SLADYTYKV
SIAGINYAK AVNNLSYTK KNFFAHHPK ILLPKDYEK FLLGAFYL
VWDPAKNML TYLPAEADI KVILAGHSK EYQDQIGCL VLIDGLSQL
QPLPNISQM RYNMAVNNL SFDNLVSTL YFHDGQNVF ILASLGFLL
APYDRVGSL DFQQIMVRL YYDLPLNEL NPDISRMIV GLSQLLPVI
APAVIESLV EHTDNPTIL YFDLFFGTI IPWSENLPD FLLNHYMTV
FYYTYGLLA APIAQNPNV ALEYIHHLF QFGGKGVEY MLIPNVDRA
SKYAFAGE LPSDQQPYV LPLNELDIL IGLEYQDQI KLEEMAKQV
TEGAGNLI YVYPLLAQG IPQGSIIGM VYFHDGQN VLKRGVYTI
LADWTNFYY QGLDNLKVI DPELQKQFA MEVVKPFI KVIAGLLRK
SLVMYYNKD KYLYAAPI AVYTFDAPG YLKMKEHKL TLNYEHMNK
KEAGVKVTL GELTGYEI QSLTPEERE KLSPDQRIF NIGYFFFKK
KSTAVLGTV NPNVLVVKK AIYAASQI RIFIYVGTE KYTDVIEKF
GAKTDDTTK KLSKQFFGD LEIPAHQI FIDETYRTK KYDDSVSTI
SQKFVDFLV GSPRFIYE LLDLAPQVP DTDRSYPVV TFNQMIKEL
QAFKDAKVN AVNNLSYTK WQIEDKHFV YIDSSLCYY DYPETQSVF
AVIESLVMY KIFDKIGVE TLGRLTQLL TQFIGLEYQ TPRAINNTL
DAKTAANDA MVRLSDGQF LYFDLFFGT KDTDRSYPV APLLVNGEL
YGVATIPTL YVYPLLAQG SINDLASLK LCYYHDLIA YIDHTNVAY
KTAAIIDGP VVQATTSAK SINDLASLK NVFNSKESF KQNGDSYGY
KAYEKEAGV TLEKLSKQF YYDLPLNEL FLLNHYMTV
AGNGAYVFG VAAGVLAAC QKVILAGHS FYLYNGDLS
AWVIPQAVK LDNLKVIEL GTDDSIIGW KSFAPLLV
NMAVNNLSY TYLSFDNLV DETVVRTV
FGTILDAGI YIDHTNVAY
NQITAVYTF MLKDKIAFL
KLRKIKTD
KLELFYETG
KIAFLGSNI
SVPRTSYLS
FGFGLSLFS
STIRSIEQV
FRKTTDNPF
TVVRTVRDS
STIRSIEQV
DGLSQLLPV
FGFGLSLFS
KLVDQGEGF
SP0024 SP1072 SP0641
Fragments Fragments Fragments
(SEQ ID NOS (SEQ ID NOS (SEQ ID NOS
170-193, 194-227, 228-264,
respectively, respectively, respectively,
in order of in order of in order of
appearance) appearance) appearance)
AIVTCMDSR GIEVEKPLY AAYAPNEVV
AQTFENEPF AEAHLLYRM AGDLRGKII
AYVALHGQL ALLNQDNMR DEIANEVWY
DDVIISGAI APPERNYLY DNYLIYGDL
FENEPFQEY AQNSYIHIL DQKEHPEKF
FMQANQAYV AVASMGTAL DSLTDRLKL
ISQQQMGTR AYLLTKTRI EAKNKNKFV
KPKTRVAIV DAAKFYHAI EGQGRNRKL
LHGQLNLPL DTALEELER EIKGAGDLR
LHVAQALGL EEYQGVPFI EPIAEGQYF
LPLKPKTRV EFLEKIAPL EVSELKPHR
MGTREIVVL EFQVLYDLL GAFFDKSKI
MQLLIESPL EHVEHLKRL GDLKWDGLI
QANQAYVAL ELSEVEMTR GEVEKNLEV
QFMQANQAY ESPLVLNDY IHFESVEEM
QLNLPLKPK GEKTPSFNV IMFIVGIFL
QQMGTREIV GLCPFHGEK IPGTLNKGI
REIVVLHHT IGDMPVQIV IRYQVFTFK
SPLIPDDVI ITMPVTKQL ISDKGGFNW
SRLHVAQAL KALLNQDNM IVSEEDFIL
TEDMIRSLV KRLTKKLVL KEIGVEEAI
VDVSDQDFL LTKTRISPI KIVVKDFAR
VSDQDFLPF LVLVYDGDK KKINFQPSL
VTEDMIRSL MRAEAHLLY KLKFVYIGK
NGPEDLAYL KVYYGNNYK
QTEEVERAW KYWQAIRAL
SEIYLMEGF LHIDNTRDF
SPHQALYDM MRFKKEDLK
VDKQVIEEI NESVVDNYL
VEMTRNKAL NEVWYAGAA
VLYDLLGQY NINDIVDGL
VPFIEAVQI QYLLKDNII
WYQVLAQDL SPRQQGAGL
YLMEGFMDV SRSKTLGGY
SSLKNTKVL
TAAVILAAY
WTELPAMGY
Thus, in some aspects, this application provides an immunogenic fragment of an antigen described herein. The fragments, in some instances, are close in size to the full-length polypeptide or the polypeptide of Table 1 or 2. For example, they may lack at most one, two, three, four, five, ten, or twenty amino acids from one or both termini. In certain embodiments, the polypeptide is 100-500 amino acids in length, or 150-450, or 200-400, or 250-250 amino acids in length. In some embodiments, the polypeptide is 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acids in length. The fragments described above or sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the biological activities described below, such as increasing the amount of IL-17 released by at least 1.5 fold or 2 fold or more (e.g., either as an absolute measure or relative to an immunologically inactive protein such as ovalbumin). A fragment may be used as the polypeptide in the vaccines described herein or may be fused to another protein, protein fragment or a polypeptide.
In some embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 1-13 having from 1-5, 1-10, or 1-20 amino acid residues removed from the N-terminus, C-terminus, or both. In some such embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus.
In certain aspects, this application provides immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a polypeptide of Table 1 or 2. The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof, and optionally further comprising a polypeptide having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to either of SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof. In certain embodiments, the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at least one of said polypeptides has an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to one of SEQ ID NOS: 1-10 or an immunogenic fragment thereof.
In some embodiments, one or more, e.g. two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof are provided in a mixture. In some embodiments, two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof are covalently bound to each other, e.g. as a fusion protein.
In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Table 1. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Tables 1 or 2.
In certain embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NO: 1-13. In certain such embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence consisting of any of SEQ ID NO: 1-13. In some embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising (or consisting of) any of the amino acid sequences of the polypeptides of Tables 1 and 2. Substantially, in this context, refers to less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2, or even less than 1% of the other S. pneumoniae polypeptides.
In certain embodiments, the vaccine composition induces a T H17 cell response at least 1.5-fold than that induced by a control irrelevant antigen (such as the HSV-2 protein ICP47 with the gene name US12) after contacting T H17 cells. In some embodiments, the vaccine formulation inhibits infection by S. pneumoniae in an uninfected subject. In certain embodiments, the vaccine formulation inhibits S. pneumoniae colonization in an individual. In some embodiments, the vaccine formulation inhibits S. pneumoniae symptoms.
In certain embodiments, this application provides nucleic acids encoding one or more of the polypeptides described above, such as DNA, RNA, or an analog thereof. The underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention. For instance, the DNA sequence may be codon-optimized to improve expression in a host such as E. coli, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
In certain embodiments, this application provides nucleic acids (such as DNA. RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a gene in Table 1 or 2, or a variant or portion of said gene. In certain embodiments, the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000 nucleotides in length. The nucleic acids may be used, for example, for recombinant production of the polypeptides of Tables 1 and 2, or immunogenic fragments thereof.
In some embodiments, the vaccine or immunogenic composition may comprise fusion proteins and/or fusion DNA constructs. The polypeptides described herein may be used without modification. In certain embodiments, when smaller related polypeptides are used, such as fragments or the like, and their molecular weight is less than about 5000 daltons, e.g., 1500 to 5000 daltons, modification may be useful in eliciting the desired immune response. For example, the smaller polypeptides can be conjugated to an appropriate immunogenic carrier such as tetanus toxoid, pneumolysin keyhole limpet hemocyanin or the like. In certain embodiments, the vaccine formulation comprises at least one lipidated polypeptide. Conjugation may be direct or indirect (e.g., via a linker). In other embodiments, a construct may comprise a gene or protein from Table 1 or 2 or an immunogenic fragment or variant thereof and a tag. A tag may be N-terminal or C-terminal. For instance, tags may be added to the nucleic acid or polypeptide to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein or nucleic acid. For instance, a purification tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification. Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G, thioredoxin, V5, avidin, streptavidin, BCCP, Calmodulin, Nus, S tags, lipoprotein D, and β galactosidase. Particular exemplary His tags include HHHHHH (SEQ ID NO: 32) and MSYYHHHHHH (SEQ ID NO: 33). In other embodiments, the polypeptide is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag. In some embodiments, the fused portion is short. This, in some instances, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of the polypeptide of Table 1 or 2.
B. Immunogenic Compositions
The present disclosure also provides pharmaceutical compositions containing immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides together with a pharmaceutical carrier. Antigens from S. pneumoniae were identified by screening immune cells from mice infected with S. pneumonia, or from healthy human donors. The human donors had presumably been exposed to S. pneumoniae at some point during their lifetimes, because S. pneumoniae is a very common disease and colonizing pathogen. Briefly, a library of S. pneumoniae antigens was expressed in bacteria and mixed with antigen presenting cells (APCs). The APCs, in turn, presented S. pneumoniae-derived polypeptides to lymphocytes that had been isolated from mice or from human donors. Lymphocyte responses were assayed for reactivity to S. pneumoniae. Human donors, as well as mice immunized with S. pneumoniae, produced lymphocytes specific to S. pneumoniae antigens. Thus, the present disclosure contemplates compositions of the S. pneumoniae antigens that elicit a strong immune response in immunized or infected mice or humans for counteracting infection by S. pneumoniae.
Tables 1 and 2 list the protein sequence and corresponding nucleotide sequence for S. pneumoniae antigens identified according to the screening methods described herein. The antigens were identified in screens of mouse and human T cells. In the screens of mouse T cells, the identified antigens were subjected to at least two rounds of screening: a genome-wide round to identify pools of 4 antigens that elicited an immune response, followed by a deconvolution round to individually test and identify single antigens that elicited an immune response from a pool identified in the genome-wide round. In contrast, in the screens of human T cells, two different sets of antigen pools were created, such that a polypeptide was combined with different polypeptides between the first and second pools. Consequently, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in positive pools in both the first and second sets. Table 1 lists antigens (and variants thereof) that were identified by one of the above screening methods, and were subsequently subjected to further testing in the mouse model described in Examples 5-8. Thus, compositions according to this disclosure may include one or two or more of the genes listed in Table 1 or 2, or the corresponding gene products.
An immunogenic composition may also comprise portions of said Streptococcus polypeptides, for example deletion mutants, truncation mutants, oligonucleotides, and peptide fragments. In some embodiments, the portions of said polypeptides are immunogenic. The immunogenicity of a portion of a protein is readily determined using the same assays that are used to determine the immunogenicity of the full-length protein. In some embodiments, the portion of the polypeptide has substantially the same immunogenicity as the full-length proteins. In some embodiments, the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less than that of the full-length protein (e.g., polypeptides of Tables 1 and 2). The peptide fragments may be, for example, linear, circular, or branched.
Some embodiments of the vaccine formulations and immunogenic compositions described herein include an immunogenic polypeptide (e.g., a polypeptide of Table 1 or 2) that contains a membrane translocating sequence (MTS), to facilitate introduction of the polypeptide into the mammalian cell and subsequent stimulation of the cell-mediated immune response. Exemplary membrane translocating sequences include hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of α-synuclein, β-synuclein, or γ-synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin β3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680 and 6,248,558.
In certain embodiments, an antigen (e.g., a polypeptide of Table 1 or 2) is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioabailability, or immunogenicity of the antigen. Molecules that may be covalently bound to the antigen include a carbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, ranging from molecular weights of below 300 g/mol to over 10,000,000 g/mol. PEG chains can be linear, branched, or with comb or star geometries. In some embodiments, the naturally produced form of a protein is covalently bound to a moeity that stimulates the immune system. An example of such a moeity is a lipid moeity. In some instances, lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR2, and activate the innate immune system.
C. Antibodies Specific to the Proteins of Tables 1 and 2
Another aspect disclosed herein is an antibody preparation generated against an antigenic composition (e.g., one of the proteins listed in Table 1 or 2 or an immunogenic fragment thereof). For instance, this disclosure provides combinations of two, three, four, or five antibodies each recognizing a different protein of Table 1 or 2. Any of a variety of antibodies are included. Such antibodies include, e.g., polyclonal, monoclonal, recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof, etc. The antibodies can be of any isotype, e.g., IgG, various IgG isotypes such as IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, etc.; and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like. In some embodiments, Fab molecules are expressed and assembled in a genetically transformed host like E. coli. A lambda vector system is available thus to express a population of Fab's with a potential diversity equal to or exceeding that of subject generating the predecessor antibody. See Huse et al. (1989), Science 246, 1275-81.
D. Components of a Vaccine or Immunogenic Composition Comprising S. pneumoniae Antigens or Antibodies Recognizing the Same
In certain embodiments, the vaccine or immunogenic composition comprises an antigen and one or more of the following: an adjuvant, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and an antibody specific to said antigen.
1. Adjuvants
The vaccine formulations and immunogenic compositions described herein may include an adjuvant. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et al., Curr. HIV Res. 1:309-20, 2003). Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), which may be, for example, particles and/or matrices, and liposomes. In contrast, immunostimulatory adjuvants are sometimes derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. An example of an organic adjuvant is cholera toxin.
Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the activation of T H1 cells or TH2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
In certain embodiments, the adjuvant induces the activation of T H17 cells. It may promote the T H17 cells to secrete IL-17. In some embodiments, an adjuvant that induces the activation of T H17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL-17 levels secreted by two populations of cells: (1) cells treated with the adjuvant and a polypeptide known to induce T H17 activation, and (2) cells treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the activation of T H17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL-17 may be measured, for example, by ELISA or Western blot. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli, or ETEC), activate a T 1117 response. Thus, in some embodiments, the adjuvant is a toxin. Cholera toxin was successfully used in the mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1 (see Examples 5-8). One form of labile toxin is produced by Intercell. Mutant derivates of labile toxin that are active as adjuvants but significantly less toxic can be used as well. Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al., “Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants” PNAS Vol. 92, pp. 1644-1648, February 1995) and LTK63 (Williams et al., “Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease” The Journal of Immunology, 2004, 173: 7435-7443), LT-G192 (Douce et al. “Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants” Infect Immun. 1999 September; 67(9):4400-6), and LTR72 (“Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.” J Exp Med. 1998 Apr. 6:187(7):1123-32).
In some embodiments, the adjuvant comprises a VLP (virus-like particle). One such adjuvant platform, Alphavirus replicons, induces the activation of T H17 cells using alphavirus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Pat. Nos. 5,643,576 and 6,783,939. In some embodiments, a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.
Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a T 11 17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from gram negative bacteria, such as S. minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular. TLR4 agonists) are disclosed in Evans J T et al. “Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529.” Expert Rev Vaccines 2003 April; 2(2):219-29. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka S T et al. “E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant.” Expert Rev. Vaccines. 2007 October; 6(5):773-84.). Various TLR agonists (including TLR4 agonists) have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids. Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).
In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.
A particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs). ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline). In certain embodiments, an ISCOM is assembled together with a polypeptide or nucleic acid of interest. However, different saponin fractions may be used in different ratios. In addition, the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions). In this context, “substantially” refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%. Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A:30-5 C, such as 70 A:30 C to 75 A:5 C, 75 A:5 C to 80 A:20 C, 80 A:20 C to 85 A:15 C, 85 A:15 C to 90 A:10 C, 90 A:10 C to 95 A:5 C, or 95 A:5 C to 99 A:1 C.
In certain embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.
Adjuvants may be covalently bound to antigens. In some embodiments, the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen-presenting cells (APCs). In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al., Cancer Res 2005; 65(11), pp 4947-4954). In certain embodiments, a polypeptide described herein is presented in the context of the trivalent S. pneumoniae Pneumococcal surface adhesin A: pneumolysin derivative carrying three amino acid substitutions (W433F, D385N, and C428G) which render the molecule nontoxic but do not interfere with TLR4-mediated inflammatory properties-cell wall polysaccharide (PsaA:PdT-CPs) conjugate system described in Lu et al. (“Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide.” Infect Immun. 2009 May; 77(5):2076-83). The conjugate system is “a fusion protein of PsaA with the pneumolysin nontoxic derivative PdT and then coupled CPs to the fusion protein”. In some embodiments, one or more polypeptides described herein is used in place of PsaA in the trivalent conjugate. The trivalent conjugate system typically includes alum and is usually administered parenterally. Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.
Typically, the same adjuvant or mixture of adjuvants is present in each dose of a vaccine. Optionally, however, an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (i.e., booster shots). Alternatively, a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
2. Additional Components of a Vaccine or Immunogenic Composition
In addition to the antigens and the adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components.
In certain embodiments, the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
In certain embodiments, the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.
In certain embodiments, the vaccine formulation or immunogenic composition includes one or more surfactants such as polysorbate 80 (TWEEN® 80), Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-(1,1,3,3 Tetramethylbutyl)phenyl-polyethylene glycol (TRITON® X-100); Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN® 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL®). A surfactant can be ionic or nonionic.
In certain embodiments, the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
In certain embodiments, a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
In certain embodiments, the vaccine formulation or immunogenic composition is a controlled release formulation.
E. DNA Vaccines
In certain aspects, the vaccine comprises one of the nucleic acids disclosed herein or a nucleic acid corresponding to one of the polypeptides described herein. When a nucleic acid vaccine is administered to a patient, the corresponding gene product (such as a desired antigen) is produced in the patient's body. In some embodiments, nucleic acid vaccine vectors that include optimized recombinant polynucleotides can be delivered to a mammal (including humans) to induce a therapeutic or prophylactic immune response. The nucleic acid may be, for example, DNA, RNA, or a synthetic nucleic acid. The nucleic acid may be single stranded or double stranded.
Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes, papillomaviruses, retroviruses, etc.) can be administered directly to the mammal for transduction of cells in vivo. The nucleic acid vaccines can be formulated as pharmaceutical compositions for administration in any suitable manner, including parenteral administration.
In determining the effective amount of the vector to be administered in the treatment or prophylaxis of an infection or other condition, the physician evaluates vector toxicities, progression of the disease, and the production of anti-vector antibodies, if any. Often, the dose equivalent of a naked nucleic acid from a vector is from about 1 μg to 1 mg for a typical 70 kilogram patient, and doses of vectors used to deliver the nucleic acid are calculated to yield an equivalent amount of therapeutic nucleic acid. Administration can be accomplished via single or divided doses. The toxicity and therapeutic efficacy of the nucleic acid vaccine vectors can be determined using standard pharmaceutical procedures in cell cultures or experimental animals.
A nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, or a combination thereof. In some embodiments, the vaccine comprises one or more cloning or expression vectors; for instance, the vaccine may comprise a plurality of expression vectors each capable of autonomous expression of a nucleotide coding region in a mammalian cell to produce at least one immunogenic polypeptide. An expression vector often includes a eukaryotic promoter sequence, such as the nucleotide sequence of a strong eukaryotic promoter, operably linked to one or more coding regions. The compositions and methods herein may involve the use of any particular eukaryotic promoter, and a wide variety are known; such as a CMV or RSV promoter. The promoter can be heterologous with respect to the host cell. The promoter used may be a constitutive promoter.
A vector useful in the present compositions and methods can be circular or linear, single-stranded or double stranded and can be a plasmid, cosmid, or episome. In a suitable embodiment, each nucleotide coding region is on a separate vector; however, it is to be understood that one or more coding regions can be present on a single vector, and these coding regions can be under the control of a single or multiple promoters.
Numerous plasmids may be used for the production of nucleic acid vaccines. Suitable embodiments of the nucleic acid vaccine employ constructs using the plasmids VR1012 (Vical Inc., San Diego Calif.), pCMVI.UBF3/2 (S. Johnston. University of Texas) or pcDNA3.1 (InVitrogen Corporation, Carlsbad, Calif.) as the vector. In addition, the vector construct can contain immunostimulatory sequences (ISS), such as unmethylated dCpG motifs, that stimulate the animal's immune system. The nucleic acid vaccine can also encode a fusion product containing the immunogenic polypeptide. Plasmid DNA can also be delivered using attenuated bacteria as delivery system, a method that is suitable for DNA vaccines that are administered orally. Bacteria are transformed with an independently replicating plasmid, which becomes released into the host cell cytoplasm following the death of the attenuated bacterium in the host cell.
An alternative approach to delivering the nucleic acid to an animal involves the use of a viral or bacterial vector. Examples of suitable viral vectors include adenovirus, polio virus, pox viruses such as vaccinia, canary pox, and fowl pox, herpes viruses, including catfish herpes virus, adenovirus-associated vector, and retroviruses. Exemplary bacterial vectors include attenuated forms of Salmonella, Shigella, Edwardsiella ictaluri, Yersinia ruckerii, and Listeria monocytogenes. In some embodiments, the nucleic acid is a vector, such as a plasmid, that is capable of autologous expression of the nucleotide sequence encoding the immunogenic polypeptide.
F. Use of Vaccines
The S. pneumoniae vaccines described herein may be used for prophylactic and/or therapeutic treatment of S. pneumoniae. Accordingly, this application provides a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein. In some aspects, the method inhibits S. pneumoniae colonization in an individual. In some aspects, the method inhibits S. pneumoniae symptoms. The subject receiving the vaccination may be a male or a female, and may be a child or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal.
1. Prophylactic Use
In prophylactic embodiments, the vaccine is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae, for example by protecting against colonization, the first and necessary step in disease. Thus, in some aspects, the method inhibits infection by S. pneumoniae in a noncolonized or uninfected subject. In another aspect, the method may reduce the duration of colonization in an individual that is already colonized.
In some embodiments, the vaccine compositions of the invention confer protective immunity, allowing a vaccinated individual to exhibit delayed onset of symptoms or reduced severity of symptoms, as the result of his or her exposure to the vaccine. In certain embodiments, the reduction in severity of symptoms is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%. In particular embodiments, vaccinated individuals may display no symptoms upon contact with S. pneumoniae, do not become colonized by S. pneumoniae, or both. Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. In particular, cellular immunity may be mediated by T H1 or T H17 cells.
Essentially any individual has a certain risk of becoming infected with S. pneumoniae. However, certain sub-populations have an increased risk of infection. In some embodiments, a vaccine formulation as described herein (e.g., a composition comprising one or more polypeptides from Table 1 or 2, or nucleic acids encoding the polypeptides, or antibodies reactive with the polypeptides) is administered to patients that are immunocompromised.
An immunocompromising condition arising from a medical treatment is likely to expose the individual in question to a higher risk of infection with S. pneumoniae. It is possible to treat an infection prophylactically in an individual having the immunocompromised condition before or during treatments known to compromise immune function. By prophylactically treating with an antigenic composition (e.g., two or more antigens from Table 1 or 2, or nucleic acids encoding the antigens), or with antibodies reactive to two or more antigens from Table 1 or 2, before or during a treatment known to compromise immune function, it is possible to prevent a subsequent S. pneumoniae infection or to reduce the risk of the individual contracting an infection due to the immunocompromised condition. Should the individual contract an S. pneumoniae infection e.g., following a treatment leading to an immunocompromised condition it is also possible to treat the infection by administering to the individual an antigen composition.
The following groups are at increased risk of pneumococcal disease or its complications, and therefore it is advantageous for subjects falling into one or more of these groups to receive a vaccine formulation described herein: children, especially those from 1 month to 5 years old or 2 months to 2 years old; children who are at least 2 years of age with asplenia, splenic dysfunction or sickle-cell disease; children who are at least 2 years of age with nephrotic syndrome, chronic cerebrospinal fluid leak, HIV infection or other conditions associated with immunosuppression.
In another embodiment, at least one dose of the pneumococcal antigen composition is given to adults in the following groups at increased risk of pneumococcal disease or its complications: all persons 65 years of age; adults with asplenia, splenic dysfunction or sickle-cell disease; adults with the following conditions: chronic cardiorespiratory disease, cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDS and other conditions associated with immunosuppression (Hodgkin's disease, lymphoma, multiple myeloma, immunosuppression for organ transplantation), individuals with cochlear implants; individuals with long-term health problems such as heart disease and lung disease, as well as individuals who are taking any drug or treatment that lowers the body's resistance to infection, such as long-term steroids, certain cancer drugs, radiation therapy; Alaskan natives and certain Native American populations.
2. Therapeutic Use
In therapeutic applications, the vaccine may be administered to a patient suffering from S. pneumoniae infection, in an amount sufficient to treat the patient. Treating the patient, in this case, refers to reducing symptoms, bacterial load, or both of S. pneumoniae in an infected individual. In some embodiments, treating the patient refers to reducing the duration of symptoms or reducing the intensity of symptoms. In some embodiments, the vaccine reduces transmissibility of S. pneumoniae from the vaccinated patient. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%.
In therapeutic embodiments, the vaccine is administered to an individual post-infection. The vaccine may be administered shortly after infection, e.g. before symptoms manifest, or may be administered during or after manifestation of symptoms.
A therapeutic S. pneumoniae vaccine can reduce the intensity and/or duration symptoms of the various indications of S. pneumoniae infection. A S. pneumoniae infection can take many forms. In some cases, an infected patient develops pneumonia, acute sinusitis, otitis media (ear infection), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.
3. Assaying Vaccination Efficacy
The efficacy of vaccination with the vaccines disclosed herein may be determined in a number of ways, in addition to the clinical outcomes described above. First, one may assay IL-17 levels (particularly IL-17A) by stimulating T cells derived from the subject after vaccination. The IL-17 levels may be compared to IL-17 levels in the same subject before vaccination. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine. Alternatively (or in combination), one may assay neutrophils in the presence of T cells or antibodies from the patient for pneumococcal killing. Increased pneumococcal killing, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine. In addition, one may measure T H17 cell activation, where increased T H17 cell activation, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the vaccine. One may also measure levels of an antibody specific to the vaccine, where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased vaccine efficacy. In certain embodiments, two or more of these assays are used. For example, one may measure IL-17 levels and the levels of vaccine-specific antibody. Alternatively, one may follow epidemiological markers such as incidence of, severity of, or duration of pneumococcal infection in vaccinated individuals compared to unvaccinated individuals.
Vaccine efficacy may also be assayed in various model systems such as the mouse model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering the test vaccine to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of S. pneumoniae. In some cases, the challenge dose is sufficient to cause S. pneumoniae colonization (especially nasal colonization) in an unvaccinated animal, and in some cases the challenge dose is sufficient to cause a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals. Examples 5 and 6 show the efficacy of polypeptides of Table 1 in inhibiting S. pneumoniae nasal colonization in the mouse model.
G. Use of Immunogenic Compositions
1. Defense Against S. pneumoniae Infection
The immunogenic compositions of the present disclosure are designed to elicit an immune response against S. pneumoniae. Compositions described herein (e.g., ones comprising one or more polypeptides of Table 1 or 2, or nucleic acids encoding the polypeptides) may stimulate an antibody response or a cell-mediated immune response, or both, in the mammal to which it is administered. In some embodiments, the composition stimulates a TH1-biased CD4+ T cell response, a TH17-biased CD4+ T cell response, or a CD8+ T cell response; in the case of a single component composition, the composition may stimulate an antibody response, a TH1-biased CD4+ T cell response, TH17-biased CD4+ T cell response, and/or a CD8+ T cell response.
In certain embodiments, the composition (e.g., one comprising one or more polypeptides of Table 1 or 2, or nucleic acids encoding the polypeptides, or antibodies reactive with the peptides) includes a cytokine or nucleotide coding region encoding a cytokine such as IL-17, to provide additional stimulation to the immune system of the mammal. In certain embodiments, the composition comprises a cytokine such as IL-17.
While not wishing to be bound by theory, in some embodiments a T H17 cell response is beneficial in mounting an immune response to the compositions disclosed herein, e.g., ones comprising one or more polypeptides of Table 1 or 2. In certain embodiments, an active T H17 response is beneficial in clearing a pneumococcal infection. For instance, mice lacking the IL-17A receptor show decreased whole cell vaccine-based protection from a pneumococcal challenge (Lu et al., “Interleukin-17A mediates acquired immunity to pneumococcal colonization.” PLoS Pathog. 2008 Sep. 19; 4(9)). Furthermore, the same authors showed that the response level of IL-17A was increased in mice treated with a whole-cell vaccine.
Thus, herein is provided a method of increasing IL-17 production by administering the compositions described herein (e.g., ones comprising one or more polypeptides of Table 1 or 2) to a subject. Furthermore, this application provides a method of activating TH7 cells by administering said compositions to a subject. In certain embodiments, increased IL-17A levels result in increased pneumococcal killing by neutrophils or neutrophil-like cells, for instance by inducing recruitment and activation of neutrophils of neutrophil-like cells. In certain embodiments, this pneumococcal killing is independent of antibodies and complement. However, specific antibody production and complement activation may be useful additional mechanisms that contribute to clearing of a pneumococcal infection.
Immunogenic compositions containing immunogenic polypeptides or polynucleotides encoding immunogenic polypeptides together with a pharmaceutical carrier are also provided.
In some instances, the immunogenic composition comprises one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, such as one or more nucleic acids selected from SEQ ID Nos. 24-31. In some embodiments these nucleic acids are expressed in the immunized individual, producing the encoded S. pneumoniae antigens, and the S. pneumoniae antigens so produced can produce an immunostimulatory effect in the immunized individual.
Such a nucleic acid-containing immunostimulatory composition may comprise, for example, an origin of replication, and a promoter that drives expression of one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13. Such a composition may also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, and a polyadenylation/transcriptional termination sequence. In some instances, the nucleic acid is DNA.
H. Diagnostic Uses
This application provides, inter alia, a rapid, inexpensive, sensitive, and specific method for detection of S. pneumoniae in patients. In this respect it should be useful to all hospitals and physicians examining and treating patients with or at risk for S. pneumoniae infection. Detection kits can be simple enough to be set up in any local hospital laboratory, and the antibodies and antigen-binding portions thereof can readily be made available to all hospitals treating patients with or at risk for S. pneumoniae infection. As used herein, “patient” refers to an individual (such as a human) that either has an S. pneumoniae infection or has the potential to contract an S. pneumoniae infection. A patient may be an individual (such as a human) that has an S. pneumoniae infection, has the potential to contract an S. pneumoniae infection, who has recovered from S. pneumoniae infection, and/or an individual whose infection status is unknown.
In some embodiments, one may perform a diagnostic assay using two or more antibodies, each of which binds one of the antigens of Table 1 and 2 to detect S. pneumoniae in an individual. The instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more S. pneumoniae-specific antibodies or antigen-binding portions thereof under conditions that allow for binding of the antibody or antigen-binding portion to an epitope of S. pneumoniae; where binding indicates the presence of S. pneumoniae in the sample. In some embodiments, the binding to the biological sample is compared to binding of the same antibody to a negative control tissue, wherein if the biological sample shows the presence of S. pneumoniae as compared to the negative control tissue, the patient is identified as likely having a S. pneumoniae infection. In some cases, binding of one antibody indicates the presence of S. pneumoniae; in other cases, the binding of two or more antibodies indicates the presence of S. pneumoniae. The aforementioned test may be appropriately adjusted to detect other bacterial infections, for instance by using an antibody immunoreactive a homolog (from another bacterial species) of one of the proteins described in Table 1. In some embodiments, the antibodies raised against a S. pneumoniae protein in Table 1 or 2 will also bind the homolog in another Streptococcus species, especially if the homologs have a high percentage sequence identity.
Alternatively, one may use an antigen of Table 1 or 2 to detect anti-S. pneumoniae antibodies in an individual. The instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more S. pneumoniae-specific antigens selected from Table 1 or 2 or portions thereof under conditions that allow for binding of the antigen (or portion thereof) to any host antibodies present in the sample; where binding indicates the presence of anti-S. pneumoniae antibodies in the sample. In some embodiments, the binding to the biological sample is compared to binding of the same antigen to a negative control tissue, wherein if the biological sample shows the presence of anti-S. pneumoniae antibodies as compared to the negative control tissue, the patient is identified as likely either (1) having a S. pneumoniae infection, or (2) having had a S. pneumoniae infection in the past. In some cases, detecting one antibody indicates a current or past infection with S. pneumoniae; in other cases, detecting two or more antibodies indicates a current or past infection with S. pneumoniae. The aforementioned test may be appropriately adjusted to detect other bacterial infections, for instance by using a homolog (from another bacterial species (e.g., a Streptococcal species) of the proteins described in Table 1.
In some embodiments, the immune cell response of a mammalian cell may be quantified ex vivo. A method for such quantification comprises administering the compositions herein disclosed to a mammalian T cell ex vivo, and quantifying the change in cytokine production of the mammalian T cell in response to the composition. In these methods, the cytokine may be, for example, IL-17.
The binding of an S. pneumoniae antibody to an antigen (e.g., a polypeptide of Table 1 or 2) may be measured using any appropriate method. Such methods include ELISA (enzyme-linked immunosorbent assay), Western blotting, competition assay, and spot-blot. The detection step may be, for instance, chemiluminescent, fluorescent, or colorimetric. One suitable method for measuring antibody-protein binding is the Luminex xMAP system, where peptides are bound to a dye-containing microsphere. Certain systems, including the xMAP system, are amenable to measuring several different markers in multiplex, and could be used to measure levels of antibodies at once. In some embodiments, other systems are used to assay a plurality of markers in multiplex. For example, profiling may be performed using any of the following systems: antigen microarrays, bead microarrays, nanobarcodes particle technology, arrayed proteins from cDNA expression libraries, protein in situ array, protein arrays of living transformants, universal protein array, lab-on-a-chip microfluidics, and peptides on pins. Another type of clinical assay is a chemiluminescent assay to detect antibody binding. In some such assays, including the VITROS Eci anti-HCV assay, antibodies are bound to a solid-phase support made up of microparticles in liquid suspension, and a surface fluorometer is used to quantify the enzymatic generation of a fluorescent product.
In some embodiments, if the biological sample shows the presence of S. pneumoniae (e.g., by detecting one or more polypeptide of Table 1 or 2 or an antibody that binds one of said polypeptides), one may administer a therapeutically effective amount of the compositions and therapies described herein to the patient. The biological sample may comprise, for example, blood, semen, urine, vaginal fluid, mucus, saliva, feces, urine, cerebrospinal fluid, or a tissue sample. In some embodiments, the biological sample is an organ intended for transplantation. In certain embodiments, before the detection step, the biological sample is subject to culture conditions that promote the growth of S. pneumoniae.
The diagnostic tests herein (e.g., those that detect a polypeptide of Table 1 or 2 or an antibody that binds one of said polypeptides) may be used to detect S. pneumoniae in a variety of samples, including samples taken from patients and samples obtained from other sources. For example, the diagnostic tests may be used to detect S. pneumoniae in food, drink, or ingredients for food and drink; on objects such as medical instruments, medical devices such as cochlear implants and pacemakers, shoes, clothing, furniture including hospital furniture, and drapes including hospital drapes; or in samples taken from the environment such as plant samples. In some embodiments, the tests herein may be performed on samples taken from animals such as agricultural animals (cows, pigs, chickens, goats, horses and the like), companion animals (dogs, cats, birds, and the like), or wild animals. In certain embodiments, the tests herein may be performed on samples taken from cell cultures such as cultures of human cells that produce a therapeutic protein, cultures of bacteria intended to produce a useful biological molecule, or cultures of cells grown for research purposes.
This disclosure also provides a method of determining the location of a S. pneumoniae infection in a patient comprising: (a) administering a pharmaceutical composition comprising a labeled S. pneumoniae antibody or antigen-binding portion thereof to the patient, and (b) detecting the label, wherein binding indicates a S. pneumoniae infection in a particular location in the patient. Such a diagnostic may also comprise comparing the levels of binding in the patient to a control. In certain embodiments, the method further comprises, if the patient has a S. pneumoniae infection, treating the infection by administering a therapeutically effective amount of a S. pneumoniae-binding antibody or antigen-binding portion thereof to the patient. In certain embodiments, the method further comprises, if the patient has a S. pneumoniae infection, treating the infection by administering a therapeutically effective amount of a S. pneumoniae protein of Table 1, or immunogenic portion thereof, to the patient. The method may further comprise determining the location and/or volume of the S. pneumoniae in the patient. This method may be used to evaluate the spread of S. pneumoniae in the patient and determine whether a localized therapy is appropriate.
In some embodiments, the anti-S. pneumoniae antibodies described herein may be used to make a prognosis of the course of infection. In some embodiments, the anti-S. pneumoniae antibodies herein may be detected in a sample taken from a patient. If antibodies are present at normal levels, it would indicate that the patient has raised an immune response against anti-S. pneumoniae. If antibodies are absent, or present at reduced levels, it would indicate that the patient is failing to raise a sufficient response against anti-S. pneumoniae, and a more aggressive treatment would be recommended. In some embodiments, antibodies present at reduced levels refers to antibodies that are present at less than 50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies typical in a patient with a normal immune system. Antibodies may be detected by affinity for any of the antigens described herein (e.g., those in Table 1 and/or 2), for example using ELISA.
In some embodiments, detection of specific S. pneumoniae antigens (e.g., those in Table 1 and/or 2) may be used to predict the progress and symptoms of S. pneumoniae infection in a patient. It will be understood by one of skill in the art that the methods herein are not limited to detection of S. pneumoniae. Other embodiments include the detection of related bacteria including bacteria with proteins homologous to the proteins described in Table 1 or 2. Such related bacteria include, for example, other strains of Streptococcus.
I. Doses and Routes of Administration
1. Dosage Forms, Amounts, and Timing
The amount of antigen in each vaccine or immunogenic composition dose is selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses. Preferably, the dose is without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific antigen is employed. Generally, it is expected that a dose will comprise 1-1000 μg of protein, in some instances 2-100 μg, for instance 4-40 μg. In some aspects, the vaccine formulation comprises 1-1000 pig of the polypeptide and 1-250 μg of the adjuvant. In some embodiments, the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immunocompromised status) of a subject. When present, typically an adjuvant will be present in amounts from 1 μg-250 μg per dose, for example 50-150 μg, 75-125 μg or 100 μg.
In some embodiments, only one dose of the vaccine is administered to achieve the results described above. In other embodiments, following an initial vaccination, subjects receive one or more boost vaccinations, for a total of two, three, four or five vaccinations. Advantageously, the number is three or fewer. A boost vaccination may be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months. It may be advantageous to administer split doses of vaccines which may be administered by the same or different routes.
The vaccines and immunogenic compositions described herein may take on a variety of dosage forms. In certain embodiments, the composition is provided in solid or powdered (e.g., lyophilized) form; it also may be provided in solution form. In certain embodiments, a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.
In some embodiments, the composition will be administered in a dose escalation manner, such that successive administrations of the composition contain a higher concentration of composition than previous administrations. In some embodiments, the composition will be administered in a manner such that successive administrations of the composition contain a lower concentration of composition than previous administrations.
In therapeutic applications, compositions are administered to a patient suffering from a disease in an amount sufficient to treat the patient. Therapeutic applications of a composition described herein include reducing transmissibility, slowing disease progression, reducing bacterial viability or replication, or inhibiting the expression of proteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the levels at which they would occur in individuals who are not treated with the composition.
In prophylactic embodiments, compositions are administered to a human or other mammal to induce an immune response that can inhibit the establishment of an infectious disease or other condition. In some embodiments, a composition may partially block the bacterium from establishing an infection.
In some embodiments, the compositions are administered in combination with antibiotics. This co-administration is particularly appropriate when the pharmaceutical composition is administered to a patient who has recently been exposed (or ia suspected of having been recently exposed) to S. pneumoniae. Many antibiotics are used to treat pneumococcal infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and vancomycin. The appropriate antibiotic may be selected based on the type and severity of the infection, as well as any known antibiotic resistance of the infection (Jacobs M R “Drug-resistant Streptococcus pneumoniae: rational antibiotic choices” Am J Med. 1999 May 3; 106(5A):19S-25S).
2. Routes of Administration
The vaccine formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled) or they can be administered by topical application. In some embodiments, the route of administration is intramuscular. In other embodiments, the route of administration is subcutaneous. In yet other embodiments, the route of administration is mucosal. In certain embodiments, the route of administration is transdermal or intradermal Certain routes of administration are particularly appropriate for vaccine formulations and immunogenic compositions comprising specified adjuvants. In particular, transdermal administration is one suitable route of administration for S. pneumoniae vaccines comprising toxins (e.g. cholera toxin or labile toxin); in other embodiments, the administration is intranasal.
Vaccines formulated with Alphavirus replicons may be administered, for example, by the intramuscular or the subcutaneous route. Vaccines comprising Monophosphory Lipid A (MPL), Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA) are suitable (inter alia) for intramuscular and subcutaneous administration. A vaccine comprising resiquimod may be administered topically or subcutaneously, for example.
3. Formulations
The vaccine formulation or immunogenic composition may be suitable for administration to a human patient, and vaccine or immunogenic composition preparation may conform to USFDA guidelines. In some embodiments, the vaccine formulation or immunogenic composition is suitable for administration to a non-human animal. In some embodiments, the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules. The vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects. In some embodiments, the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments. In some embodiments, the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other vaccine or immunogenic composition components may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation. In other embodiments, the pyrogens may be inactivated or destroyed prior to administration to a patient. Raw materials for vaccines, such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be sterile, and each lot of the vaccine may be tested for sterility. Thus, in certain embodiments the endotoxin levels in the vaccine fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non-intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water.
In certain embodiments, the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein. In certain embodiments, the desired molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture). In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). In some embodiments, the vaccine or immunogenic composition comprising purified subunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of protein from host cells in which the subunit proteins were expressed, relative to the amount of purified subunit. In some embodiments, the desired polypeptides are substantially free of nucleic acids and/or carbohydrates. For instance, in some embodiments, the vaccine or immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type are present in the preparation (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
It is preferred that the vaccine or immunogenic composition has low or no toxicity, within a reasonable risk-benefit ratio. In certain embodiments, the vaccine or immunogenic composition comprises ingredients at concentrations that are less than LD50 measurements for the animal being vaccinated. LD50 measurements may be obtained in mice or other experimental model systems, and extrapolated to humans and other animals. Methods for estimating the LD50 of compounds in humans and other animals are well-known in the art. A vaccine formulation or immunogenic composition, and any component within it, might have an LD50 value in rats of greater than 100 g/kg, greater than 50 g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg. A vaccine formulation or immunogenic composition that comprises a toxin such as botulinum toxin (which can be used as an adjuvant) should contain significantly less than the LD50 of botulinum toxin.
The formulations suitable for introduction of the vaccine formulations or pharmaceutical composition vary according to route of administration. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. In the case of adoptive transfer of therapeutic T cells, the cells can be administered intravenously or parenterally.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
The antigens, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptides or packaged nucleic acids with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the polypeptides or packaged nucleic acids with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
J. Preparation and Storage of Vaccine Formulations and Immunogenic Compositions
The S. pneumoniae vaccines and immunogenic compositions described herein may be produced using a variety of techniques. For example, a polypeptide may be produced using recombinant DNA technology in a suitable host cell. A suitable host cell may be bacterial, yeast, mammalian, or other type of cell. The host cell may be modified to express an exogenous copy of one of the relevant polypeptide genes. Typically, the gene is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence. In some embodiments, the promoter is inducible or repressible. Other regulatory sequences may provide for secretion or excretion of the polypeptide of interest or retention of the polypeptide of interest in the cytoplasm or in the membrane, depending on how one wishes to purify the polypeptide. The gene may be present on an extrachromosomal plasmid, or may be integrated into the host genome. One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and may be desirable. For instance, the nucleic acid may be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same. In some embodiments, the gene is codon-optimized to improve expression in a particular host. The nucleic acid may be produced, for example, by PCR or by chemical synthesis.
Once a recombinant cell line has been produced, a polypeptide may be isolated from it. The isolation may be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).
In a further aspect of the present disclosure, there is provided a method of manufacture comprising mixing one or more polypeptides or an immunogenic fragment or variant thereof with a carrier and/or an adjuvant.
In some embodiments, antigens for inclusion the vaccine formulations and immunogenic compositions may be produced in cell culture. One method comprises providing one or more expression vectors and cloning nucleotides encoding one or more polypeptides selected from polypeptides having an amino acid sequence of Table 1 or Table 2, then expressing and isolating the polypeptides.
The immunogenic polypeptides described herein, and nucleic acid compositions that express the polypeptides, can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal. For example, packs or dispenser devices that contain one or more unit dosage forms are provided. Typically, instructions for administration of the compounds will be provided with the packaging, along with a suitable indication on the label that the compound is suitable for treatment of an indicated condition, such as those disclosed herein.
V. EXAMPLES Example 1
Antigen Identification and Pooled Murine Screens
Each open reading frame predicted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major histocompatibility complex (MHC). Each construct was then expressed in E. coli, and full-length expression validated by a surrogate assay that identifies the tag in the context of MHC. The screen is described in more detail in International Application WO 2010/002993. In order to facilitate screening the large library, the library was pooled such that four induced library clones were present in each well. In order to screen T cells from mice immunized against S. pneumoniae, an aliquot of the pooled library was added to peritoneal-derived macrophages. The macrophages were allowed to bind the tagged S. pneumoniae antigens via the MHC. After 2 hr at 37° C., the macrophages were washed with PBS. The macrophages were then fixed with 1% paraformaldehyde for 15 min and washed extensively with PBS. 105 T cells were added to each well in 200 μL of RP-10 media. The T cells had previously been isolated from mice that had been immunized 2 times with killed S. pneumoniae bacteria with cholera toxin adjuvant. The assay plates were incubated for 72 hrs at 37° C. The amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay. The threshold for a positive result was set at two standard deviations above the mean of all samples.
Example 2
Deconvolution of the Positive Murine Pools
A secondary screen was used to determine which antigen(s) out of the four clones in each well induced the positive response observed in the pooled screen described in Example 1. All the clones in each positive pool were pulsed individually onto peritoneal macrophages in duplicate wells. T cells isolated from immunized mice from the same genetic background as the initial screen were used to screen the pulsed macrophages using the IL-17 assay described in Example 1. Individual antigens that induced an average response in the duplicate wells greater than two standard deviations above the mean of negative control samples were considered positive responses. The library plasmids present in these positive clones were sequenced to confirm the identity of the antigen. The antigens SP_1574, SP_1655, SP_2106, SP_0148. SP_1473, SP_0605, SP_1177, SP_0335, SP_0906, SP_1828, SP_2157, SP_1229, SP_1128, SP_1836. SP_1865, SP_0904, SP_0882, SP_0765. SP_1634, SP_0418, SP_1923, SP_1313, SP_0775. SP_0314, SP_0912, SP_0159, SP_0910. SP_2148, SP_1412, SP_0372, SP_1304. SP_2002, SP_0612, SP_1988, SP_0484, SP_0847, SP_1527, SP_0542, SP_0441, SP_0350, SP_0014, SP_1965, SP_0117, and SP_2108 were confirmed using this method.
Example 3
Antigen Identification and Pooled Human Screens
CD4+ T cells and CD14+ monocytes were isolated from peripheral blood acquired from human donors. The monocytes were differentiated into dendritic cells by culturing them in GM-CSF and IL-4 containing media, essentially as described in Tedder T F and Jansen P J (1997 “Isolation and generation of human dendritic cells.” Current Protocols in Immunology Supp 23: 7.32.1-7.32.16). After five days in culture, the dendritic cells were seeded into 384 well plates. The CD4+ T cells were expanded in culture to ensure sufficient quantities.
Each open reading frame predicted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major histocompatibility complex (MHC). Each construct was then expressed in E. coli, and full-length expression validated by a surrogate assay that identifies the tag in the context of MHC. In order to facilitate screening the large library, the library was pooled such that four induced library clones were present in each well. In order to screen the human T cells, an aliquot of the pooled library was added to the seeded dendritic cells in 384-well plates. After 2 hr at 37° C., the dendritic cells were fixed with 1% paraformaldehyde for 15 min and washed extensively with phosphate buffer and lysine buffer. 40,000 of the CD4+ T cells in 70 μL of RP-10 media were added to each well of a 384-well plate. The assay plates were incubated for 3 days at 37° C. The amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay. In different iterations of the screen, the threshold for a positive result was set at two standard deviations above the mean of all samples, two standard deviations above the mean of negative controls, or 1.78 times the median absolution deviation of the data set. Positive pools were then deconvoluted as described in Example 4.
Example 4
Deconvolution of the Positive Human Pools
For all antigens, deconvolution was performed by comparing the results of two pool screens. In this method, two different sets of pools were prepared, so that a polypeptide was with three different polypeptides between the first and second pools. Consequently, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in positive pools in both the first and second sets. In this deconvolution method, a pool was identified as positive if it was at least 1.78 times the median absolution deviation of the data set.
An antigen was identified as a positive hit if it was positive in at least two repeated secondary screens. The antigens SP2108, SP0641, SP1393, SP0024, SP0641.1, SP1072, SP1384 and SP2032 were identified using the above approach.
Example 5
SP2108, SP0148 and SP1634 Polypeptides
The SP2108 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 7) and SP1634 polypeptide (see Table 2) were formulated as vaccine compositions using 4 μg of the polypeptide in combination with 1 μg cholera toxin. For combinations, 4 μg of each polypeptide was used. The compositions were administered intranasally to C57BU/6 mice three times, one week apart. The subjects were then allowed to rest for 3 weeks, and bled at that time for immunogenicity. For this assay, heparinized whole blood was collected from the retrograde orbital sinus. The total PBMC were stimulated with either killed whole cells (WCC) or a combination of the three polypeptides in round bottomed tubes for three days. The supernatants were then harvested and evaluated by ELISA for IL-17 levels. Cholera toxin alone (CT) or an unrelated antigen from HSV (003) were used as negative controls. Results are shown in FIGS. 1 and 2. The subjects were allowed to rest an additional 2 weeks, at which time they were challenged with intranasal administration of S. pneumoniae. The subjects were sacrificed a week later, and the number of colony-forming units (CFU) was counted from nasal washes. Results are shown in FIG. 3.
Example 6
SP0882 and SP0314 Polypeptides
This example used the same protocols as Example 5, except that only two doses of the vaccine composition were administered. In these experiments, the SP0882 polypeptide (SEQ ID NO: 2) and SP0314 polypeptides (see Table 2) were used in conjunction with the three polypeptides tested in Example 5. Results of the immunogenicity assay are shown in FIGS. 4 and 5. Results of the colonization assay are shown in FIG. 6.
Example 7
SP1072, SP0641N, and SP0024 Polypeptides
This example used a protocol similar to that of Example 5, except that two doses of the vaccine composition were administered, one week apart. Four weeks after the last immunization, the mice were challenged intranasally with live type 6B S. pneumoniae. One week later the bacterial burden was assessed in each mouse by plating a nasal lavage on selective media and counting CFU. The CFU isolated from each mouse is plotted for each immunized cohort. The results are shown in FIG. 7. Statistically significant results are indicated in the figure (*=p=value<0.05).
Example 8
SP0148, SP0314, SP0882, and SP2108 Polypeptides Tested in the BALB/c Mouse
To determine whether similar immune responses were seen across different mouse genotypes, several polypeptides were administered to BALB/c mice. Using a protocol similar to that of Example 5, the mice were immunized, challenged with S. pneumoniae, and the number of CFU was recorded. The results of this experiment are shown in FIG. 8.
SEQUENCES
Potypeptide Sequences
SEQ ID NO: 1
SP0024
>gi|14971488|gb|AAK74215.1| conserved hypothetical
protein Streptococcus pneumoniae TIGR4
MSYFEQFMQANQAYVALHGQLNLPLKPKTRVAIVTCMDSRLHVAQALGLA
LGDAHILRNAGGRVTEDMIRSLVISQQQMGTREIVVLHHTDCGAQTFENE
PFQEYLKEELGVDVSDQDFLPFQDIEESVREDMQLLIESPLIPDDVIISG
AIYNVDTGSMTVVEL
SEQ ID NO: 2
SP0882
>gi|14972356|gb|AAK75009.1| conserved hypothetical
protein (Streptococcus pneumoniae TIGR4)
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDG
QNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWK
FQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSL
GGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFI
YVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKQSG
AIHSEIPWSENLPDCLRFFAEKW
SEQ ID NO: 3
SP0882N
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSPYVVYFHDG
QNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWK
RQESPIPGQQFGGKGVEYAEFVMEVVKPFI
SEQ ID NO: 4
SP0882 with exogenous leader
MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERR
VRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNP
DISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEV
VKPF

Claims (34)

We claim:
1. A vaccine formulation comprising:
(i) a pharmaceutically acceptable carrier,
(ii) an effective amount of an adjuvant,
(iii) a first polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID 6 or SEQ ID 7, and
(iv) a second polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID 9 or SEQ ID 10.
2. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 9.
3. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 10.
4. The vaccine formulation of claim 1, wherein the polypeptide is conjugated to an immunogenic carrier.
5. The vaccine formulation of claim 1, which comprises at least one lipidated polypeptide.
6. The vaccine formulation of claim 1, wherein the adjuvant is an agonist of toll-like receptors (TLRs).
7. The vaccine formulation of claim 1, wherein the adjuvant is alum.
8. The vaccine formulation of claim 1, wherein the vaccine formulation inhibits infection by Streptococcus pneumoniae in an uninfected subject.
9. The vaccine formulation of claim 1, wherein the vaccine formulation inhibits Streptococcus pneumoniae colonization in an individual.
10. The vaccine formulation of claim 1, wherein the vaccine formulation inhibits Streptococcus pneumoniae symptoms.
11. A method for treating a subject suffering from or susceptible to Streptococcus pneumoniae infection, comprising administering an effective amount of a vaccine formulation according to claim 1.
12. The method of claim 11, wherein the method inhibits infection by Streptococcus pneumoniae in an uninfected subject.
13. The method of claim 11, wherein the method inhibits Streptococcus pneumoniae colonization in an individual.
14. The method of claim 11, wherein the method inhibits Streptococcus pneumoniae symptoms.
15. The method of claim 11, wherein the method treats a subject with one dose.
16. The method of claim 11, wherein the method treats a subject within three doses.
17. The method of claim 11, wherein the subject is a human.
18. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 6.
19. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 7.
20. A pharmaceutical composition comprising:
(i) a pharmaceutically acceptable carrier,
(ii) an effective amount of an adjuvant,
(iii) a first polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID 6 or SEQ ID 7, and
(iv) a second polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID 9 or SEQ ID 10.
21. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 6.
22. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 7.
23. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 9.
24. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 10.
25. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 6.
26. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 7.
27. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 9.
28. The pharmaceutical composition of claim 20, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 10.
29. The pharmaceutical composition of claim 20, wherein the polypeptide is conjugated to an immunogenic carrier.
30. The pharmaceutical composition of claim 20, which comprises at least one lipidated polypeptide.
31. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 6.
32. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 7.
33. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 9.
34. The vaccine formulation of claim 1, which comprises a polypeptide having an amino acid sequence consisting of SEQ ID NO: 10.
US16/133,590 2009-06-29 2018-09-17 Vaccines and compositions against Streptococcus pneumoniae Active 2030-12-09 US11207375B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/133,590 US11207375B2 (en) 2009-06-29 2018-09-17 Vaccines and compositions against Streptococcus pneumoniae

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US22154109P 2009-06-29 2009-06-29
US24061609P 2009-09-08 2009-09-08
US24059809P 2009-09-08 2009-09-08
US31626710P 2010-03-22 2010-03-22
US12/826,084 US10105412B2 (en) 2009-06-29 2010-06-29 Vaccines and compositions against Streptococcus pneumoniae
US16/133,590 US11207375B2 (en) 2009-06-29 2018-09-17 Vaccines and compositions against Streptococcus pneumoniae

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/826,084 Division US10105412B2 (en) 2009-06-29 2010-06-29 Vaccines and compositions against Streptococcus pneumoniae

Publications (2)

Publication Number Publication Date
US20190298795A1 US20190298795A1 (en) 2019-10-03
US11207375B2 true US11207375B2 (en) 2021-12-28

Family

ID=43449681

Family Applications (2)

Application Number Title Priority Date Filing Date
US12/826,084 Active 2031-05-29 US10105412B2 (en) 2009-06-29 2010-06-29 Vaccines and compositions against Streptococcus pneumoniae
US16/133,590 Active 2030-12-09 US11207375B2 (en) 2009-06-29 2018-09-17 Vaccines and compositions against Streptococcus pneumoniae

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US12/826,084 Active 2031-05-29 US10105412B2 (en) 2009-06-29 2010-06-29 Vaccines and compositions against Streptococcus pneumoniae

Country Status (15)

Country Link
US (2) US10105412B2 (en)
EP (1) EP2448592B1 (en)
JP (1) JP5931724B2 (en)
KR (1) KR101748453B1 (en)
CN (2) CN102548572A (en)
AU (1) AU2010273708B2 (en)
BR (1) BRPI1011919A2 (en)
CA (1) CA2803061A1 (en)
IL (1) IL217166A0 (en)
IN (1) IN2012DN00791A (en)
MX (1) MX2012000158A (en)
NZ (1) NZ597858A (en)
RU (1) RU2580299C2 (en)
SG (2) SG10201403702SA (en)
WO (1) WO2011008548A1 (en)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI1011919A2 (en) 2009-06-29 2017-09-26 Childrens Medical Center Streptococcus pneumoniae vaccines and compositions
EP3165531B1 (en) 2010-03-12 2020-11-25 Children's Medical Center Corporation Immunogenic composition, vaccine, and uses thereof
PL2656070T3 (en) * 2010-12-21 2017-01-31 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Determination of the efficacy of a live recombinant anti-mycobacterial vaccination
AU2012207089B2 (en) 2011-01-20 2016-04-14 Children's Medical Center Corporation Vaccines and compositions against Streptococcus pneumoniae
AR085698A1 (en) 2011-03-09 2013-10-23 Glaxosmithkline Llc INHIBITORS OF THE PEPTIDE DESFORMILASA
BR112013028887B1 (en) 2011-05-11 2020-10-06 Children's Medical Center Corporation IMMUNOGENIC COMPOSITION PRESENTING MULTIPLE ANTIGENS, AND METHODS AND USES OF THE SAME
JP6379041B2 (en) * 2012-01-20 2018-08-22 ジェノセア バイオサイエンシーズ， インコーポレイテッド Fusion antigen vaccines and compositions for Streptococcus pneumoniae
WO2014018904A1 (en) * 2012-07-26 2014-01-30 Genocea Biosciences, Inc. Fused antigen vaccines and compositions against streptococcus pneumoniae
CA2900008A1 (en) 2013-02-07 2014-08-14 Children's Medical Center Corporation Protein antigens that provide protection against pneumococcal colonization and/or disease
US20150079132A1 (en) * 2013-09-17 2015-03-19 Path Evoking protection against streptotoccus pneumoniae incorporating b-cell and t-cell pneumococcal protein antigens and pneumococcal polysaccharides delivered concomitantly
WO2015161218A1 (en) * 2014-04-18 2015-10-22 Children's Medical Center Corporation Vaccine adjuvant compositions
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
HRP20220573T1 (en) 2017-01-20 2022-06-10 Pfizer Inc. Immunogenic compositions for use in pneumococcal vaccines
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
AU2018243910A1 (en) 2017-03-28 2019-11-14 The Children's Medical Center Corporation A multiple antigen presenting system (MAPS)-based staphylococcus aureus vaccine, immunogenic composition, and uses thereof
BR112019027387A8 (en) 2017-06-23 2022-12-06 Univ Maryland IMMUNOGENIC COMPOSITIONS
CA3111459A1 (en) 2018-09-12 2020-03-19 Affinivax, Inc. Multivalent pneumococcal vaccines
RU2728938C1 (en) * 2020-05-06 2020-08-03 Федеральное государственное бюджетное учреждение науки "Научный центр биомедицинских технологий Федерального медико-биологического агентства" (ФГБУН НЦБМТ ФМБА России) Use of dalargin for production of drug preparations for preventing pneumonia

Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5371197A (en) 1991-09-24 1994-12-06 Merck & Co., Inc. Protein-dimeric polysaccharide conjugate vaccine
US5623057A (en) 1991-01-28 1997-04-22 Merck & Co., Inc. Pneumococcal polysaccharide conjugate vaccine
US5643576A (en) 1994-05-27 1997-07-01 The University Of North Carolina At Chapel Hill Method of inducing an immune response with a live Venezuelan Equine Encephalitis virus expressing a heterologous immunogen
WO1997037026A1 (en) 1996-04-02 1997-10-09 Smithkline Beecham Corporation Novel compounds
WO1998018931A2 (en) 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
WO1998050554A2 (en) 1997-05-06 1998-11-12 Human Genome Sciences, Inc. Enterococcus faecalis polynucleotides and polypeptides
WO1998059071A1 (en) 1997-06-20 1998-12-30 Human Genome Sciences, Inc. Lyme disease vaccines
US5882885A (en) 1997-07-17 1999-03-16 Smithkline Beecham Corporation Glycogen phosphorylase
US5994101A (en) 1997-07-18 1999-11-30 Smithkline Beecham Corporation DNA encoding gidA1 polypeptides
WO2000006738A2 (en) 1998-07-27 2000-02-10 Microbial Technics Limited NUCLEIC ACIDS AND PROTEINS FROM $i(STREPTOCOCCUS PNEUMONIAE)
US6248558B1 (en) 1998-03-31 2001-06-19 Vanderbilt University Sequence and method for genetic engineering of proteins with cell membrane translocating activity
WO2002077021A2 (en) 2001-03-27 2002-10-03 Chiron Srl. Streptococcus pneumoniae proteins and nucleic acids
WO2002083855A2 (en) 2001-04-16 2002-10-24 Wyeth Holdings Corporation Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
EP1328543A2 (en) 2000-10-27 2003-07-23 CHIRON S.p.A. Nucleic acids and proteins from streptococcus groups a & b
US6617156B1 (en) 1997-08-15 2003-09-09 Lynn A. Doucette-Stamm Nucleic acid and amino acid sequences relating to Enterococcus faecalis for diagnostics and therapeutics
WO2003082183A2 (en) 2002-04-02 2003-10-09 Ben-Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
US20040029129A1 (en) 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms
US6699703B1 (en) * 1997-07-02 2004-03-02 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
WO2004018646A2 (en) 2002-08-26 2004-03-04 Chiron Corporation Conserved and specific streptococcal genomes
WO2004020609A2 (en) 2002-08-30 2004-03-11 Tufts University Streptococcus pneumoniae antigens for diagnosis, treatment and prevention of active infection
US6783939B2 (en) 2000-07-07 2004-08-31 Alphavax, Inc. Alphavirus vectors and virosomes with modified HIV genes for use in vaccines
WO2004092209A2 (en) 2003-04-15 2004-10-28 Intercell Ag S. pneumoniae antigens
WO2005014630A2 (en) 2003-08-08 2005-02-17 Chiron Srl Streptococcus pneumoniae knockout mutants
US6887480B1 (en) 1999-06-10 2005-05-03 Medimmune, Inc. Streptococcus pneumoniae proteins and vaccines
US6951732B2 (en) 1999-12-23 2005-10-04 Vmax Ltd. Virulence genes, proteins, and their use
EP1185297B1 (en) 1999-06-10 2006-05-10 MedImmune, Inc. Streptococcus pneumoniae proteins and vaccines
WO2006069200A2 (en) 2004-12-22 2006-06-29 Novartis Vaccines And Diagnostics Inc. Group b streptococcus
WO2006084467A1 (en) 2005-02-11 2006-08-17 Ace Biosciences A/S Surface-located streptococcus pneumoniae polypeptides
WO2007106407A2 (en) 2006-03-10 2007-09-20 Wyeth Microarray for monitoring gene expression in multiple strains of streptococcus pneumoniae
US7314974B2 (en) 2002-02-21 2008-01-01 Monsanto Technology, Llc Expression of microbial proteins in plants for production of plants with improved properties
WO2008081014A2 (en) 2007-01-04 2008-07-10 Glaxosmithkline Biologicals S.A. Process for manufacturing vaccines
WO2008119358A2 (en) 2007-03-30 2008-10-09 Ace Biosciences A/S Surface-located streptococcus pneumoniae polypeptides for use in vaccine compositions
WO2008146164A2 (en) 2007-05-25 2008-12-04 Novartis Ag Streptococcus pneumoniae pilus antigens
WO2009011971A2 (en) 2007-05-18 2009-01-22 The Govt. Of The U.S.A. As Represented By The Secretary Of The Dept. Of Health And Human Services, Centers For Disease Control And Prevention Primers and probes for the detection of streptococcus pneumoniae
WO2009016515A2 (en) 2007-08-01 2009-02-05 Novartis Ag Compositions comprising pneumococcal antigens
WO2009033742A2 (en) 2007-09-14 2009-03-19 Katholieke Universiteit Leuven Streptococcus pneumoniae vaccines
US20090221499A1 (en) 2007-12-12 2009-09-03 National Health Research Institutes Lipidating Sequences and Use thereof for Producing Lipidated Proteins in E. Coli
US20090252756A1 (en) 2002-04-02 2009-10-08 Yaffa Mizrachi-Nebenzahl Protein-based streptococcus pneumoniae vaccines
US20100015168A1 (en) 2006-06-09 2010-01-21 Novartis Ag Immunogenic compositions for streptococcus agalactiae
WO2010064243A1 (en) 2008-12-03 2010-06-10 Protea Vaccine Technologies Ltd. GLUTAMYL tRNA SYNTHETASE (GtS) FRAGMENTS
US20100166785A1 (en) 2007-12-07 2010-07-01 National Health Research Institutes Adjuvants
US20100247547A1 (en) 2007-05-29 2010-09-30 Chen Dong Modulation of the th-17 cell mediated immune responses
US20100303849A1 (en) 2009-06-02 2010-12-02 National Health Research Institutes Lipidated Vaccine against Dengue Virus Infection
US20100322953A1 (en) 2009-06-22 2010-12-23 National Health Research Institutes Lipidated Tumor-Associated Antigens and Immunotherapeutic Compositions
WO2010150242A2 (en) 2009-06-25 2010-12-29 Protea Vaccine Technologies Ltd. Immunogenic streptococcus pneumoniae peptides and peptide-multimers
WO2011008548A1 (en) 2009-06-29 2011-01-20 Genocea Biosciences, Inc. Vaccines and compositions against streptococcus pneumoniae
WO2011064781A1 (en) 2009-11-30 2011-06-03 Ben-Gurion University Of The Negev Research And Development Authority S. pneumoniae adhesins
US20110135686A1 (en) * 2008-03-14 2011-06-09 Sanofi Pasteur Biologics Co. Replication-Defective Flavivirus Vaccines and Vaccine Vectors
WO2011067758A2 (en) 2009-12-02 2011-06-09 Protea Vaccine Technologies Ltd. Immunogenic fragments and multimers from streptococcus pneumoniae proteins
US20110246740A1 (en) 2010-04-06 2011-10-06 Hitachi, Ltd. Management method and management apparatus
WO2011128892A2 (en) 2010-04-12 2011-10-20 Protea Vaccine Technologies Ltd. Modified forms of pneumococcal surface immunogenic protein b (psipb)
US20110283062A1 (en) 2010-05-14 2011-11-17 Hitachi, Ltd. Storage apparatus and data retaining method for storage apparatus
US20120189649A1 (en) 2011-01-20 2012-07-26 Todd Gierahn Vaccines and Compositions Against Streptococcus Pneumoniae

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2728927C (en) 2008-07-01 2018-02-13 Genocea Biosciences, Inc. Antigen screening system

Patent Citations (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5623057A (en) 1991-01-28 1997-04-22 Merck & Co., Inc. Pneumococcal polysaccharide conjugate vaccine
US5371197A (en) 1991-09-24 1994-12-06 Merck & Co., Inc. Protein-dimeric polysaccharide conjugate vaccine
US5643576A (en) 1994-05-27 1997-07-01 The University Of North Carolina At Chapel Hill Method of inducing an immune response with a live Venezuelan Equine Encephalitis virus expressing a heterologous immunogen
WO1997037026A1 (en) 1996-04-02 1997-10-09 Smithkline Beecham Corporation Novel compounds
EP0942983A2 (en) 1996-10-31 1999-09-22 Human Genome Sciences, Inc. $i(STREPTOCOCCUS PNEUMONIAE) ANTIGENS AND VACCINES
WO1998018930A2 (en) 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Streptococcus pneumoniae antigens and vaccines
US7056510B1 (en) 1996-10-31 2006-06-06 Human Genome Sciences, Inc. Streptococcus pneumoniae SP036 polynucleotides, polypeptides, antigens and vaccines
US20100221287A1 (en) 1996-10-31 2010-09-02 Human Genome Sciences, Inc. Streptococcus pneumoniae Polynucleotides and Sequences
US6573082B1 (en) 1996-10-31 2003-06-03 Human Genome Sciences, Inc. Streptococcus pneumoniae antigens and vaccines
WO1998018931A2 (en) 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
US7141418B2 (en) 1996-10-31 2006-11-28 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
US20040029118A1 (en) 1996-10-31 2004-02-12 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
US20070154986A1 (en) 1996-10-31 2007-07-05 Human Genome Sciences, Inc. Streptococcus pneumoniae Polynucleotides and Sequences
WO1998050554A2 (en) 1997-05-06 1998-11-12 Human Genome Sciences, Inc. Enterococcus faecalis polynucleotides and polypeptides
WO1998059071A1 (en) 1997-06-20 1998-12-30 Human Genome Sciences, Inc. Lyme disease vaccines
US6699703B1 (en) * 1997-07-02 2004-03-02 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
US20080175854A1 (en) 1997-07-02 2008-07-24 Genome Therapeutics, Inc. Nucleic acid and amino acid sequences relating to streptococcus pneumoniae for diagnostics and therapeutics
US7378514B2 (en) 1997-07-02 2008-05-27 sanofi pasteur limited/sanofi pasteur limitée Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
US20050136404A1 (en) 1997-07-02 2005-06-23 Doucette-Stamm Lynn A. Nucleic acid and amino acid sequences relating to streptococcus pneumoniae for diagnostics and therapeutics
US20090215149A1 (en) 1997-07-02 2009-08-27 Lynn Doucette-Stamm Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
US7338786B2 (en) 1997-07-02 2008-03-04 Sanofi Pasteur Limited Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
US5882885A (en) 1997-07-17 1999-03-16 Smithkline Beecham Corporation Glycogen phosphorylase
US6235285B1 (en) 1997-07-17 2001-05-22 Smithkline Beecham Corporation Glycogen phosphorylase from streptococcus pneumoniae
US5994101A (en) 1997-07-18 1999-11-30 Smithkline Beecham Corporation DNA encoding gidA1 polypeptides
US6617156B1 (en) 1997-08-15 2003-09-09 Lynn A. Doucette-Stamm Nucleic acid and amino acid sequences relating to Enterococcus faecalis for diagnostics and therapeutics
US6248558B1 (en) 1998-03-31 2001-06-19 Vanderbilt University Sequence and method for genetic engineering of proteins with cell membrane translocating activity
US6432680B1 (en) 1998-03-31 2002-08-13 Vanderbilt University Sequence and method for genetic engineering of proteins with cell membrane translocating activity
WO2000006738A2 (en) 1998-07-27 2000-02-10 Microbial Technics Limited NUCLEIC ACIDS AND PROTEINS FROM $i(STREPTOCOCCUS PNEUMONIAE)
EP1185297B1 (en) 1999-06-10 2006-05-10 MedImmune, Inc. Streptococcus pneumoniae proteins and vaccines
US6887480B1 (en) 1999-06-10 2005-05-03 Medimmune, Inc. Streptococcus pneumoniae proteins and vaccines
US7132107B2 (en) 1999-06-10 2006-11-07 Medimmune, Inc. Streptococcus pneumoniae proteins and vaccines
US6951732B2 (en) 1999-12-23 2005-10-04 Vmax Ltd. Virulence genes, proteins, and their use
US6783939B2 (en) 2000-07-07 2004-08-31 Alphavax, Inc. Alphavirus vectors and virosomes with modified HIV genes for use in vaccines
EP1328543A2 (en) 2000-10-27 2003-07-23 CHIRON S.p.A. Nucleic acids and proteins from streptococcus groups a & b
US20060210580A1 (en) 2000-10-27 2006-09-21 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A & B
US20060210579A1 (en) 2000-10-27 2006-09-21 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A & B
EP2278008A2 (en) 2001-03-27 2011-01-26 Novartis Vaccines and Diagnostics S.r.l. Streptococcus pneumoniae proteins and nucleic acids
US20050020813A1 (en) 2001-03-27 2005-01-27 Vega Masignani Streptococcus pneumoniae proteins and nucleic acids
WO2002077021A2 (en) 2001-03-27 2002-10-03 Chiron Srl. Streptococcus pneumoniae proteins and nucleic acids
EP1630230A2 (en) 2001-03-27 2006-03-01 Chiron SRL Streptococcus pneumoniae proteins and nucleic acids
JP2005503119A (en) 2001-03-27 2005-02-03 カイロン ソチエタ ア レスポンサビリタ リミタータ STREPTOCOCCUSPNEUMONIAE protein and nucleic acid
US20120128707A1 (en) 2001-03-27 2012-05-24 J. Craig Venter Institute Streptococcus pneumoniae proteins and nucleic acids
EP1572868A2 (en) 2001-04-16 2005-09-14 Wyeth Holdings Corporation Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
US20100221288A1 (en) 2001-04-16 2010-09-02 Wyeth Holdings Corporation Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
US20040110181A1 (en) 2001-04-16 2004-06-10 Zagursky Robert John Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
WO2002083855A2 (en) 2001-04-16 2002-10-24 Wyeth Holdings Corporation Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
US7384775B2 (en) * 2001-04-16 2008-06-10 Wyeth Streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
US20040029129A1 (en) 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms
US7314974B2 (en) 2002-02-21 2008-01-01 Monsanto Technology, Llc Expression of microbial proteins in plants for production of plants with improved properties
US20090252756A1 (en) 2002-04-02 2009-10-08 Yaffa Mizrachi-Nebenzahl Protein-based streptococcus pneumoniae vaccines
WO2003082183A2 (en) 2002-04-02 2003-10-09 Ben-Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
US20050196415A1 (en) 2002-04-02 2005-09-08 Yaffa Mizrachi Nebenzahl Protein-based Streptococcus pneumoniae vaccines
US20090148470A1 (en) 2002-04-02 2009-06-11 Ben-Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
US7504110B2 (en) 2002-04-02 2009-03-17 Ben-Gurion University Of The Negev Research And Development Authority Protein-based Streptococcus pneumoniae vaccines
EP2275128A2 (en) 2002-04-02 2011-01-19 Ben Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
WO2004018646A2 (en) 2002-08-26 2004-03-04 Chiron Corporation Conserved and specific streptococcal genomes
US20070053924A1 (en) 2002-08-26 2007-03-08 Herve Tettelin Conserved and specific streptococcal genomes
WO2004020609A2 (en) 2002-08-30 2004-03-11 Tufts University Streptococcus pneumoniae antigens for diagnosis, treatment and prevention of active infection
US20100260790A1 (en) 2003-04-15 2010-10-14 Intercell Ag S. Pneumoniae Antigens
US20060263846A1 (en) 2003-04-15 2006-11-23 Intercell Ag S. penumoniae antigens
EP2311987A1 (en) 2003-04-15 2011-04-20 Intercell AG S. pneumoniae antigens
WO2004092209A2 (en) 2003-04-15 2004-10-28 Intercell Ag S. pneumoniae antigens
US7635487B2 (en) 2003-04-15 2009-12-22 Intercell Ag S. pneumoniae antigens
JP2007525157A (en) 2003-04-15 2007-09-06 インターツェル・アクチェンゲゼルシャフト Streptococcus pneumoniae antigen
US20070184443A1 (en) 2003-08-08 2007-08-09 Chiron Srl Streptococcus pneumoniae knockout mutants
WO2005014630A2 (en) 2003-08-08 2005-02-17 Chiron Srl Streptococcus pneumoniae knockout mutants
WO2006069200A2 (en) 2004-12-22 2006-06-29 Novartis Vaccines And Diagnostics Inc. Group b streptococcus
WO2006084467A1 (en) 2005-02-11 2006-08-17 Ace Biosciences A/S Surface-located streptococcus pneumoniae polypeptides
US20090202528A1 (en) 2005-02-11 2009-08-13 Thomas Kofoed Surface-Located Streptococcus Pneumoniae Polypeptides
EP1855717A1 (en) 2005-02-11 2007-11-21 Ace Biosciences A/S Surface-located streptococcus pneumoniae polypeptides
WO2007106407A2 (en) 2006-03-10 2007-09-20 Wyeth Microarray for monitoring gene expression in multiple strains of streptococcus pneumoniae
US20110177960A1 (en) 2006-03-10 2011-07-21 Ellen Murphy Microarray for monitoring gene expression in multiple strains of Streptococcus pneumoniae
US20100015168A1 (en) 2006-06-09 2010-01-21 Novartis Ag Immunogenic compositions for streptococcus agalactiae
US20100143399A1 (en) 2007-01-04 2010-06-10 Glaxosmithkline Biologicals Process for manufacturing vaccines
WO2008081014A2 (en) 2007-01-04 2008-07-10 Glaxosmithkline Biologicals S.A. Process for manufacturing vaccines
WO2008119358A2 (en) 2007-03-30 2008-10-09 Ace Biosciences A/S Surface-located streptococcus pneumoniae polypeptides for use in vaccine compositions
WO2009011971A2 (en) 2007-05-18 2009-01-22 The Govt. Of The U.S.A. As Represented By The Secretary Of The Dept. Of Health And Human Services, Centers For Disease Control And Prevention Primers and probes for the detection of streptococcus pneumoniae
WO2008146164A2 (en) 2007-05-25 2008-12-04 Novartis Ag Streptococcus pneumoniae pilus antigens
US20100247547A1 (en) 2007-05-29 2010-09-30 Chen Dong Modulation of the th-17 cell mediated immune responses
WO2009016515A2 (en) 2007-08-01 2009-02-05 Novartis Ag Compositions comprising pneumococcal antigens
US20100278819A1 (en) 2007-09-14 2010-11-04 Katholieke Universiteit Leuven Streptococcus pneumoniae vaccines
WO2009033742A2 (en) 2007-09-14 2009-03-19 Katholieke Universiteit Leuven Streptococcus pneumoniae vaccines
US20100166785A1 (en) 2007-12-07 2010-07-01 National Health Research Institutes Adjuvants
US20090221499A1 (en) 2007-12-12 2009-09-03 National Health Research Institutes Lipidating Sequences and Use thereof for Producing Lipidated Proteins in E. Coli
US20110135686A1 (en) * 2008-03-14 2011-06-09 Sanofi Pasteur Biologics Co. Replication-Defective Flavivirus Vaccines and Vaccine Vectors
WO2010064243A1 (en) 2008-12-03 2010-06-10 Protea Vaccine Technologies Ltd. GLUTAMYL tRNA SYNTHETASE (GtS) FRAGMENTS
US20100303849A1 (en) 2009-06-02 2010-12-02 National Health Research Institutes Lipidated Vaccine against Dengue Virus Infection
US20100322953A1 (en) 2009-06-22 2010-12-23 National Health Research Institutes Lipidated Tumor-Associated Antigens and Immunotherapeutic Compositions
US20120100172A1 (en) 2009-06-25 2012-04-26 Michael Tal Immunogenic streptococcus pneumoniae peptides and peptide-multimers
WO2010150242A2 (en) 2009-06-25 2010-12-29 Protea Vaccine Technologies Ltd. Immunogenic streptococcus pneumoniae peptides and peptide-multimers
WO2011008548A1 (en) 2009-06-29 2011-01-20 Genocea Biosciences, Inc. Vaccines and compositions against streptococcus pneumoniae
US20110020386A1 (en) 2009-06-29 2011-01-27 Todd Gierahn Vaccines and compositions against streptococcus pneumoniae
US10105412B2 (en) 2009-06-29 2018-10-23 Genocea Biosciences, Inc. Vaccines and compositions against Streptococcus pneumoniae
WO2011064781A1 (en) 2009-11-30 2011-06-03 Ben-Gurion University Of The Negev Research And Development Authority S. pneumoniae adhesins
WO2011067758A2 (en) 2009-12-02 2011-06-09 Protea Vaccine Technologies Ltd. Immunogenic fragments and multimers from streptococcus pneumoniae proteins
US20110246740A1 (en) 2010-04-06 2011-10-06 Hitachi, Ltd. Management method and management apparatus
WO2011128892A2 (en) 2010-04-12 2011-10-20 Protea Vaccine Technologies Ltd. Modified forms of pneumococcal surface immunogenic protein b (psipb)
WO2011141968A1 (en) 2010-05-14 2011-11-17 Hitachi, Ltd. Storage apparatus and data retaining method for storage apparatus
US20110283062A1 (en) 2010-05-14 2011-11-17 Hitachi, Ltd. Storage apparatus and data retaining method for storage apparatus
US20120189649A1 (en) 2011-01-20 2012-07-26 Todd Gierahn Vaccines and Compositions Against Streptococcus Pneumoniae

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
Briles, D.E et al., Intranasal Immunization of Mice with a Mixture of the Pneumococcal Proteins PSAA and PSPA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae, Infection and Immunity, 68(2): 796-800 (2000).
Briles, D.E. et al., The potential for using protein vaccines to protect against otitis media casued by Streptococcus pneumoniae, Vaccine, 19: S87-S95 (2001).
Brown, J.S. et al., Immunization with Components of Two Iron Uptake ABC Transporter Protests Mice against Systemic Streptococcus pneumoniae Infection, Infection and Immunity, 69(11): 6702-6706 (2001).
Burgess et al. (J of Cell Biology, 1990 vol. 111, pp. 2129-2138) (Year: 1990). *
Cao et al., Enhanced protection against pneumococcal infection elicited by immunization with the combination of PspA, PspC, and CIpP, Vaccine, 25(27): 4996-5005 (2007).
Database UniParc, F5MIE7, retrieved from internet May 28, 2013 (Jul. 27, 2011).
Database UniParc, F9HBT0, retrieved from internet May 28, 2013 (Oct. 19, 2011).
Database UniParc, F9PYR1, retrieved from internet May 28, 2013 (Oct. 19, 2011).
Database UniParc, G6J1F1, retrieved from internet May 28, 2013 (Jan. 25, 2012).
Database UniParc, G6KAH8, retrieved from internet May 28, 2013 (Jan. 25, 2012).
Database UniParc, G6U2X1, retrieved from internet May 28, 2013 (Jan. 25, 2012).
Database UniParc, G6VVK1, retrieved from internet May 28, 2013 (Jan. 25, 2012).
Database UniprotKB, Lipidation, retrieved from internet May 28, 2013 (Feb. 17, 2011).
Ding, F. et al., Genome evolution driven by host adaptations results in a more virulent and antimicrobial-resistant Streptococcus pneumoniae serotype 14, BMC Genomics,10:158 (2009).
Dopazo, J. et al., Annotated Draft Genomic Sequence from a Streptococcus pneumoniae Type 19F Clinical Isolate, Microb. Drug Resist., 7(2):99-125 (2001).
Douce, G. et al. Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants, Infect Immun., 67(9):4400-6 (1999).
Douce, G. et al., Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants, PNAS, vol. 92: 1644-1648 (1995).
Evans, J.T. et al., Enhancement of antigen-specific immunity via the TLR4 ligands MPLTM adjuvant and Ribi.529, Expert Rev., Vaccines, 2(2); 219-229 (2003).
Extended European Search Report for EP 10800303.9, 12 pages (dated Mar. 27, 2013).
Giefing et al., Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies, The Journal of Experimental Medicine, 205(1):117-131 (2008).
Giuliani et al. Mucosal adjuvanticity and immunogenicity of L TR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity, J Exp Med., 6:187(7):1123-32 (1998).
Grubaugh, D. et al., Proteins as T Cell Antigens: Methods for High-Throughput Identifications, Vaccine (2013).
Hoskins, J. et al., Genome of the Bacertium Streptococcus pneumoniae Strain R6., J Bacteriol, 183(19): 5709-5717 (2001).
Huse et al., Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda, Science, 246: 1275-1281 (1989).
International Preliminary Report on Patentability for PCT/US12/22127, dated Jul. 23, 2013, 5 pages.
International Preliminary Report on Patentability for PCT/US12/22128, dated Jul. 23, 2013, 6 pages.
International Search Report for PCT/US12/22127, dated May 15, 2012, 4 pages.
International Search Report for PCT/US12/22128, dated May 21, 2012, 4 pages.
International Search Report for PCT/US13/22309, dated May 24, 2013, 4 pages.
International Search Report of PCT/US13/29907, dated Jun. 20, 2013, 3 pages.
Ishizaka S.T. et al. E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant, Expert Rev. Vaccines, 6(5):773-84. (2007).
Jedrzejas, M.J., Unveiling molecular mechanisms of bacterial surface proteins: Streptococcus pneumoniae as a model organism for structural studies, Cell. Mol. Life Sci., 64(21): 2799-2822 (2007).
Lanie, J.A et al., Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison with That of Unencapsulated Laboratory Strain R6, J Bacteriol., 189(1): 38-51 (2007).
Lu et al. Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide, Infect Immun., 77(5): 2076-83 (2009).
Lu et al., Interleukin-17A mediates acquired immunity to pneumococcal colonization, PLoS Pathogens, 4(9): e1000159 1-11 (2008).
Morsczeck, C. et al., Streptococcus pneumoniae: proteomics of surface proteins for vaccine development, European Society of Clinical Microbiology and Infectious Diseases, CMI, 14(1): 74-81 (2007).
Rudikoff et al (Proc Natl Acad Sci USA 1982 vol. 79 p. 1979). (Year: 1979). *
Shah, P. et al., Immunization with Polyamine Transport Protein PotD Protects Mice against Systemic Infection with Streptococcus pneumoniae, Infection and Immunity 74(10): 5888-5892 (2006).
Singh et al., Advances in Vaccine Adjuvants for Infectious Diseases, Current HIV Research, 1:309-20 (2003).
Tedder et al. Isolation and generation of human dendritic cells, Current Protocols in Immunology, Supp23: 7.32.1-7.32.16 (1997).
Tettelin H et al.: Complete Genome Sequence of a Virulent Isolate of Streptococcus pneumoniae, Science, 293(5529): 498-506 (2001).
Wiezmann et al. Use of a Whole Genome Approach to Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection, Infection and Immunity, 69(3): 1593-1598 (2001).
Williams, A.E. et al., Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (L TK63) Provides Generic Protection against Lung Infectious Disease, The Journal of Immunology, 173: 7435-7443 (2004).
Written Opinion for PCT/US12/22127, dated May 15, 2012, 7 pages.
Written Opinion for PCT/US12/22128, dated May 21, 2012, 8 pages.
Written Opinion for PCT/US13/22309, dated May 24, 2013, 12 pages.
Written Opinion of PCT/US13/29907, dated Jun. 20, 2013, 4 pages.
Wu, Y. et al., Hsp70-Like Protein 1 Fusion Protein Enhances Induction of Carcinoembryonic Antigen-Specific CDS+ CTL Response by Dendritic Cell Vaccine, Cancer Res., 65(11 ), 4947-4954 (2005).
Yamamoto, M. et al., Oral Immunization with PSPA Elicits protective humoral immunity against Streptococcus pneumoniae infection, Infection and Immunity, 65(2): 640-644 (1997).

Also Published As

Publication number Publication date
IL217166A0 (en) 2012-02-29
KR101748453B1 (en) 2017-06-16
EP2448592A4 (en) 2013-04-24
EP2448592A1 (en) 2012-05-09
JP2012532134A (en) 2012-12-13
NZ597858A (en) 2014-01-31
IN2012DN00791A (en) 2015-06-26
US10105412B2 (en) 2018-10-23
AU2010273708B2 (en) 2015-10-01
CA2803061A1 (en) 2011-01-20
BRPI1011919A2 (en) 2017-09-26
AU2010273708A8 (en) 2012-06-14
KR20120093141A (en) 2012-08-22
AU2010273708A1 (en) 2012-02-23
US20190298795A1 (en) 2019-10-03
WO2011008548A8 (en) 2012-03-08
CN107126557A (en) 2017-09-05
US20110020386A1 (en) 2011-01-27
RU2580299C2 (en) 2016-04-10
WO2011008548A1 (en) 2011-01-20
SG177402A1 (en) 2012-02-28
MX2012000158A (en) 2012-06-08
CN102548572A (en) 2012-07-04
JP5931724B2 (en) 2016-06-08
RU2011153032A (en) 2013-08-10
SG10201403702SA (en) 2014-09-26
EP2448592B1 (en) 2020-06-17

Similar Documents

Publication Publication Date Title
US11207375B2 (en) Vaccines and compositions against Streptococcus pneumoniae
US10188717B2 (en) Vaccines and compositions against Streptococcus pneumoniae
JP2017160238A (en) Fused antigen vaccine and composition against streptococcus pneumoniae
US20150079132A1 (en) Evoking protection against streptotoccus pneumoniae incorporating b-cell and t-cell pneumococcal protein antigens and pneumococcal polysaccharides delivered concomitantly
NZ614460B2 (en) Vaccines and compositions against streptococcus pneumoniae

Legal Events

Date Code Title Description
FEPP Fee payment procedure

Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

FEPP Fee payment procedure

Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

AS Assignment

Owner name: CHILDREN'S MEDICAL CENTER CORPORATION, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MALLEY, RICHARD;REEL/FRAME:049213/0751

Effective date: 20190510

Owner name: CHILDREN'S MEDICAL CENTER CORPORATION, MASSACHUSET

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MALLEY, RICHARD;REEL/FRAME:049213/0751

Effective date: 20190510

Owner name: GENOCEA BIOSCIENCES, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GIERAHN, TODD;REEL/FRAME:049213/0728

Effective date: 20190513

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS

STPP Information on status: patent application and granting procedure in general

Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED

STCF Information on status: patent grant

Free format text: PATENTED CASE